JP2009523738A - Modified polymer - Google Patents

Abstract

The present invention is a polymer with controlled end group stoichiometry, comprising a surface layer, at least one sub-surface layer, and a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof. And a polymer comprising at least two end groups comprising a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer (in this case, end group stoichiometry is defined as end group stoichiometry) Number and type).

Description

Field of Invention

  The present invention relates to macromolecules, particularly dendrimers that can control their surface architecture so that the proportion of certain topological isomers is concentrated, their production, and their use. In particular, the polymer has two or more surface groups, and at least one of the surface groups can be pharmaceutically active.

Background of the Invention

  The identification of new compounds for use in pharmaceutical formulations is an important part of the search for treatments with higher reliability and effectiveness. Of equal importance, however, is the development and modification of known compounds that reduce the risks associated with new drug candidates and significantly reduce the development and cost of bringing the drug to clinical development.

  Many drugs fail in clinical trials because their physical properties (especially solubility) make formulation difficult, or because the therapeutic index is small and it causes toxic effects at high drug concentrations that occur immediately after dosing To do. Other disadvantages include low absorption, low bioavailability, instability, and inability to target the drug once administered, systemic side effects due to inability to control its biodistribution, metabolism and renal or hepatic clearance. is there. Similarly, some products currently on the market can be improved with respect to such problems.

  Several approaches have been attempted to improve the properties of pharmaceutical compounds, including the formulation of pharmaceutical agents into liposomes, micelles or polymeric micelle formulations, as well as the covalent attachment of pharmaceutical agents to hydrophilic polymer backbones.

  The ideal property modifier features a well-defined structure that allows precise control of absorption, distribution, metabolism and excretion (ADME) characteristics (also known as pharmacokinetics) of the compound in question. And preferably the ability to hold more than one compound per agent or construct. The toxicity of the compound in question can be improved by controlling the release of the compound from the drug or construct so that the body is only exposed to therapeutic plasma concentrations of the compound.

  In recent years, dendritic polymers have been increasingly applied to biotechnology and pharmaceutical applications. Dendritic polymers are a special type of polymer having a densely branched structure characterized by a higher concentration of functional groups per unit molecular volume than ordinary polymers. There are four polymeric subclasses that are classified based on the relative degree of structural control present in each dendritic architecture: random hyperbranched polymers; dendrigraft polymers; dendritic motifs; and dendrimers. In particular, dendrimers are promising as new scaffolds for drug delivery due to their unique properties such as high degree of branching, multivalency, spherical architecture and well-defined molecular weight. Research on the design and synthesis of biocompatible dendrimers and their application to many areas of bioscience, including drug delivery, has increased over the past decade.

The potential usefulness of dendritic polymers, both as drug delivery vectors and as pharmaceutically active substances, has attracted increasing interest in recent years ^1,18 . However, the literature is packed with reports of synthetic schemes, for example for dendrimer assembly, a description of dendrimer-drug interactions and drug loading efficiencies, and an in vitro evaluation of the increasing interaction between dendrimers and cell lines. although it has ^few, there is little information describes the basic pharmacokinetic and metabolic fate of the dendrimer.

The interaction between dendrimers and intestinal tissue has also been the subject of several ^studies4-11 , but the permeability and cytotoxicity trends associated with surface charge and surface functionality have been established but are absorbed into the systemic circulation. Relatively few studies have described the fate of dendrimers after they were made. Among these few studies, Gillies et al. Investigated the pharmacokinetics of PEGylated “bow-tie” polyester dendrimers, and the mechanism of dendrimer clearance strongly depends on the molecular weight and flexibility of the complex ¹² , Kobayashi et al. Investigated the effect of structural changes on the biodistribution of dendrimers designed to facilitate complexation of heavy metals or antibodies (and hence application to bioimaging). ^13-17 . But so far, the specific systemic pharmacokinetics of polylysine dendrimers have not been described in detail.

  In addition, it remains a challenge to produce dendrimers that circulate in the blood for a time sufficient to accumulate at the target site, but at the same time can be eliminated from the body at an appropriate rate to avoid long-term accumulation. is there. In addition, it is still difficult to predict the tissue localization of dendrimers in advance, and more research is needed to determine the effect of peripheral dendritic groups on these properties. Another area that needs to be studied is the release of drugs from dendrimers. The steric hindrance associated with high-density spherical dendritic architecture makes it difficult to engineer enzymatically cleavable bonds.

  Accordingly, it is an object of the present invention to overcome or at least mitigate one or more of the difficulties and / or disadvantages associated with the prior art.

Summary of the Invention

As a first aspect, the present invention provides a polymer having controlled end group stoichiometry, comprising a surface layer, at least one sub-surface layer, and the remainder of a pharmaceutically active agent, derivative or precursor thereof. A first end group that is a group, and a polymer comprising at least two end groups including a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer (in this case, the end group) Stoichiometry refers to the number and type of end groups.

  In yet another embodiment, the polymer further exhibits a controlled topology (where the topology is a certain end group in terms of its connection to the surface layer or sub-surface layer of the polymer). And the relationship to another terminal group).

  In preferred embodiments, the second end group can comprise polyethylene glycol (PEG) or polyethyloxazoline (eg, PEOX).

  In yet another embodiment, the second end group is selected from one or more of the following: a moiety that modifies the plasma half-life of the pharmaceutically active agent and / or macromolecule; one or more cell types or tissues A moiety that facilitates targeting of a pharmaceutically active agent and / or macromolecule to a type; and a moiety that facilitates incorporation of a pharmaceutically active agent and / or macromolecule into one or more cell types or tissue types.

  In preferred embodiments, the second end group is selected such that the half-life of the pharmaceutically active agent and / or macromolecule is increased.

  The polymer is preferably a dendrimer, more preferably a lysine dendrimer.

  The polymers according to this aspect of the invention can be utilized in a variety of applications, as described below, where the ability to control end group stoichiometry and / or topology provides consistent pharmacokinetic properties to pharmaceutically active agents. This is advantageous.

  As used herein and in the claims, the singular forms “a”, “an”, and “the” are intended to include a plurality of referents unless the context clearly indicates otherwise. Thus, for example, reference to “a macromolecule” includes one or more of those polymers.

  As used herein and in the claims, the term “comprises” (or grammatical variations thereof) is equivalent to the term “includes” and is intended to indicate the presence of other elements or features. Do not interpret it as excluded.

  As used herein and in the claims, the term “topology” refers to the relationship between one end group and another end group in terms of their connection to a surface layer or subsurface layer of polymers. Means.

  As used herein and in the claims, the term “topological isomer” shall mean a polymer having a certain topology.

  As used herein and in the claims, the term “surface” shall mean a layer of generational building units having surface amines that can react with “end groups” or “capping” groups.

  As used herein and in the claims, the term “subsurface” shall mean one or more layers below the surface layer.

  As used herein and in the claims, the term “surface amine” is intended to mean any surface-reactive amine group of a dendrimer.

  As used herein and in the claims, the term “end group stoichiometry” shall mean the composition (number and type) of end groups on the surface of the polymer.

  As used herein and in the claims, the term “generation building unit” is intended to mean a repeating unit that forms a dendrimer framework, such as lysine or lysine analogs in the case of lysine dendrimers.

  As used herein and in the claims, the term “dendritic motif” is intended to mean a discontinuous segment of a macromolecule. The dendritic motif is released when one of the polymer branches is cleaved at the bond connecting one of the generation building units or core reactive amines to the carboxyl of the attached generation building unit. Become. The carboxyl of the dendritic motif corresponds to a unique point or apex where, in the process of synthesizing the polymer of the present invention, the dendritic motif is attached to the growing polymer core. .

  Any polymer can be suitable for use in the present invention. The macromolecule can be selected from one or more dendritic polymers including arborol, dendrigrafts, PAMAM dendrimers, lysine dendrimers and the like.

  The preparation of dendrimer polymers is well known, for example, U.S. Pat. Nos. 4,289,872 and 4,410,688 (describing dendrimer polymers based on layers of lysine units), U.S. Pat. Nos. 4,507,466, 4,558,120. No. 4,568,737 and No. 4,587,329, which describe dendrimer polymers based on other units, including polyamidoamines or PAMAM dendrimer polymers. The dendrimer polymers disclosed in these US patents are used in aqueous formulations such as surface modifiers, metal chelators, demulsifiers or oil / water emulsions, wet strength agents in paper manufacture, and paints. It is described as being suitable for uses such as drugs for modifying viscosity. US Pat. Nos. 4,289,872 and 4,410,688 also suggest that dendrimer polymers based on lysine units can be used as a base for manufacturing pharmaceutical formulations.

  In the following, the present invention is described in detail with respect to dendrimers (especially those based on polylysine).

  One important determinant of dendrimer efficacy in any given application is the nature of the polymer surface. Applicants have surprisingly determined the absorption, distribution, metabolism and excretion (ADME) properties of certain pharmaceutical compounds by conjugating them to dendrimers in a well-defined and controlled process. I found a technique to modify. More specifically, modification of dendrimer size and / or surface functionality can regulate drug excretion (clearance), distribution and metabolic (absorption and reabsorption) properties. The process of the present invention makes it possible to produce precise structures that can vary to meet the unique requirements of the drug. Furthermore, multiple molecules can be attached to the dendrimer in a controlled manner to increase drug loading and accommodate a wider variety of therapies. Drug loading can be further modified by changing the number of generations of dendrimers.

Previous studies have suggested that uncapped ³ H-labeled poly-L-lysine dendrimers are rapidly metabolized to free lysine after intravenous administration. Surprisingly, however, PEGylation of dendrimers has been shown to reduce dendrimer recognition by proteolytic enzymes as well as serum proteins and suppress phagocytic clearance, resulting in increased plasma circulation time. Furthermore, PEGylation can increase the hydrodynamic volume of the dendrimer and consequently reduce renal clearance.

For example, the inventors have found that the plasma half-life and degree of urinary exclusion of ³ H-labeled PEGylated lysine dendrimers are dependent on molecular weight. Despite the fact that small dendrimer complexes showed signs of interaction with plasma components leading to the generation of high molecular weight species, large PEGylated dendrimers (ie,> 20 kD) compared to small dendrimers (ie, <20 kD) 30 kD) was removed relatively slowly from plasma into urine. The elimination of these complexes was rapid and only complete dendrimers were recovered in the urine. Furthermore, the present inventors determined that the size of the PEG group attached to the surface of the dendrimer determines whether the dendrimer can avoid uptake by the intranet system when the pharmaceutically active ingredient and the PEG group are attached to the surface of the dendrimer. We also observed stagnation. Thus, it is clear that increasing the size by some means does not necessarily result in extending the plasma lifetime of the dendrimer. Large dendrimers were found to accumulate in the liver and spleen. However, this occurred over a long period of time and the accumulated amount was less than 10% of the dose.

  In yet another preferred embodiment, the PEG or polyethyloxazoline end groups may comprise about 25% to 75%, more preferably about 25% to 50% of the end groups on the dendrimer. Increasing the relative size of individual PEG or polyethyloxazoline groups can maintain the required plasma life span and avoid liver uptake. The percentage of PEG or polyethyloxazoline groups and / or the size of PEG or polyethyloxazoline groups can be modified and adapted to suit different pharmaceutically active agents.

  In a preferred embodiment, the PEG groups are relatively monodisperse and are selected from a molecular weight range of 200 to 10,000 daltons, more preferably the PEG groups are selected from a molecular weight range of 500 to 5,000 daltons.

  PEGylation can also improve the solubility of the compound and thus promote the solubility of the inherently insoluble drug conjugated to the surface of the dendrimer.

  Thus, the present invention engineered drugs with high toxicity and / or low solubility to allow controlled release of such drugs so that long-term drug concentrations are maintained at plasma levels that are therapeutic but not toxic. Providing a means to get a promising vehicle.

  Dendrimers carrying two different end groups can be produced as different topological isomers (in this case, the topology is one end group different from the other in terms of its connection to the surface and subsurface layers). Represents the relationship with two end groups). Dendrimers bearing two or more different end groups are disclosed in AU2005905908, the entire disclosure of which is hereby incorporated by reference. The way each topological isomer interacts with a complex system can vary. Therefore, it may be advantageous to be able to control the surface distribution of different end groups for different applications.

  The ability to concentrate molecules with the same topology in dendritic polymer samples is as desirable as enrichment of specific stereoisomers in organic materials has been shown to be particularly desirable for biological applications right. Thus, one topology isomer in a polymer may be more effective than another topology isomer in a given application.

  Prior art methods for producing dendrimers having two or more different end groups involve synthesis using random surface functionalization methods.

  The first prior art approach uses a substoichiometric amount of the first active end group in an attempt to cap only half of the reactive terminal amine moiety (surface amine) on the surface of the polymer. The remaining surface amine can then be reacted, and in this second reaction, an excess amount of the second active end group can be used to complete the reaction. This approach results in a statistical distribution of products with different end group stoichiometry resulting from the first stage of the reaction, and little or no control over the topology of the two different end groups.

  Also, in the second prior art approach, both end groups can be reacted simultaneously with a reactive surface amine moiety. In such an approach, it would be possible to adjust the stoichiometry of each end group to account for the different reactivity of each end group, but there are more than two types of reactions with deprotected nitrogen groups. Generational building units or more than one end group can be utilized, so intermolecular variability will still occur, so the probable outcome of each reaction can only be described by a statistical distribution, here But the reaction topology results are not controlled.

  Compared to prior art random surface functionalization, the abundance of dendritic moieties with precisely specified end groups is at least 2 times higher than their abundance in randomly surface functionalized materials A polymer is considered "concentrated" if it is high (2 times monodispersity), preferably 4 times (4 times monodispersity). The enrichment concept illustrated for the end groups can also be applied to the surface couplets, quartets, octets, etc., detailed in AU2005905908, the contents of which are hereby incorporated by reference in their entirety. Macromolecules can be enriched for specific end group stoichiometry and / or topology.

  When the end group stoichiometry enrichment reaches a maximum level, each macromolecule will have end groups of the same composition (number and type). If the concentration level is not so great, for example 20% enriched, this is taken to mean that 20% of the macromolecules have end groups of the same composition (number and type).

  As another option, this concentration can be specified by comparing the composition with a randomly functionalized polymeric composition. For example, if the random method gives 5% of the polymer a specific surface composition, the enrichment constitutes an increase in the polymer with a specific end group composition (number and type) above this 5% level. Will be. Thus, a 2-fold enrichment would mean that 10% of the macromolecule showed its specific end group composition.

  The simplest type of topology enrichment is at the couplet level. The dendrimer composition may be fully enriched at the end group level, but not fully enriched at the caplet level. This is because the same end groups can be grouped into couplets in several ways. For example, in any of FIGS. 9.1-9.5, the dendrimer contains 16 terminal A groups and 16 terminal B groups. However, there are 8 (AA) and 8 (BB) couplets in FIGS. 9.1-9.4, whereas in FIG. 9.5 there are 16 (AB) couplets. Higher order topological enrichment is enrichment at the quartet, octet and 16-tet levels, which is detailed in AU2005905908.

In another embodiment, a polymer comprising at least one lysine or lysine analog dendritic motif having a surface layer and at least one sub-surface layer comprising:
A first terminal group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and Macromolecules comprising one end group (where end group stoichiometry refers to the number and type of end groups) are provided.

  In yet another embodiment of this aspect of the invention, the polymer further exhibits a controlled topology (where the topology is in terms of its connection to the surface layer or sub-surface layer of the polymer). Represents a relationship between one terminal group and another terminal group).

  In preferred embodiments, the second end group can comprise a polyethylene glycol (PEG) or polyethyloxazoline (eg, PEOX) motif.

  In yet another embodiment, the second end group is selected to modify the plasma half-life of the pharmaceutically active agent. In preferred embodiments, the second end group is selected to increase the half-life of the pharmaceutically active agent.

  In yet another embodiment, the second end group is selected to facilitate targeting and / or uptake of the pharmaceutically active agent to one or more cell types or tissue types.

In another embodiment of the invention, controlled capping group stoichiometry and formula:
Core [repeating unit] _m [surface building unit] _n [capping group 1] _p [capping group 2] _q
[Where:
The core is selected from the group consisting of lysine, or derivatives thereof, diamine compounds, triamine compounds or tetraamine compounds;
The repeat unit is selected from the group consisting of amidoamine, lysine or lysine analogs;
The surface building unit may be the same as or different from that of the building unit and is selected from amidoamine, lysine or lysine analogues;
Capping group 1 is a pharmaceutically active agent, derivative thereof, precursor thereof or residue thereof;
Capping group 2 is selected to modify the pharmacokinetics of the pharmaceutically active agent;
m represents the sum of repeating units of one or more subsurface layers of the dendrimer and is an integer from 1 to 32;
n represents the number of surface building units of one or more surface layers of the dendrimer and is an integer from 2 to 32;
p represents the number of one capping group and is an integer from 1 to 64;
q represents the number of two capping groups and is an integer from 1 to 64]
(In this case, capping group stoichiometry refers to the number and type of capping groups).

［式中、a、b、およびcは、それぞれ、0〜5（より好ましくは1〜3）の整数である］
から選択される化合物である。 The core of the dendrimer polymer can be selected from any suitable compound. Preferably, the core is selected from lysine or derivatives thereof, diamine compounds, triamine compounds, or tetraamine compounds. Most preferably, the core is benzhydrylamide-lysine (BHALys), or

[Wherein, a, b, and c are each an integer of 0 to 5 (more preferably 1 to 3)]
Is a compound selected from

［式中、aは0または1（好ましくは1）である］
の一つ以上から選択することができる。 The repeating unit according to this embodiment of the invention is preferably

[Wherein, a is 0 or 1 (preferably 1)]
You can choose from one or more.

で表されるBHALys、DAH、EDAおよびTETAからなる群より選択することができる。 In a preferred embodiment of the invention, the dendrimer has a lysine core or lysine analog core. The lysine dendritic core has the following formula:

Can be selected from the group consisting of BHALys, DAH, EDA and TETA.

Dendrimers are PAMAM polymers, such as PAMAM ₃₂ , lysine or lysine analog polymers where the repeating unit is [Lys] _Q or [Su (NPN) ₂ ] _Q , where Q is 2, 6, 14 for the divalent core. , 30 or 32, and in the case of a trivalent core, it is an integer of 3, 9, 21 or 45].

  The dendrimers of the present invention can be spread over as many generations as necessary. Preferably the dendrimer extends over 1-5 generations, more preferably 1-3 generations.

  The polymeric (especially dendrimer) pharmaceutically active agent of the present invention may comprise a water-insoluble pharmaceutical, a water-soluble pharmaceutical, a lipophilic pharmaceutical, or a mixture thereof.

Examples of pharmaceutically active agents include, but are not limited to, one or more selected from the group shown in Table 1.

  The present invention is particularly active even in very small quantities, and its long-term administration is particularly suitable for medicaments that are particularly sought to overcome the toxicity problems associated with standard doses. Examples include, but are not limited to, the antimetabolite methotrexate, the anti-mitotic drug taxol, the obesity drug zenical, the immunosuppressant drug cyclosporine, and the anti-inflammatory drug indomethacin.

  In certain embodiments, the polymers of the present invention include two or more different pharmaceutically active agents, derivatives thereof, precursors thereof, or residues thereof as end groups.

In yet another embodiment, the invention has a controlled end group stoichiometry, and
At least two end groups that are residues of different pharmaceutically active agents, derivatives or precursors thereof, and yet another end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or dendrimer A dendrimer comprising (in this case, end group stoichiometry refers to the number and type of end groups).

  The dendrimers according to this aspect of the invention can be applied in combination therapy.

  The pharmaceutically active agent can be a combination of any two or more of the categories exemplified in Table 1. Exemplary combinations include chemotherapeutic drugs; anti-inflammatory drugs and anti-arthritic drugs; anti-obesity drugs and anti-diabetic drugs; growth hormones and growth-promoting drugs; muscle relaxants and anti-inflammatory drugs; respiratory drugs and bronchodilators Microbial drugs; including but not limited to combinations of chemotherapeutic drugs and vitamins. More specific combinations are described in the examples.

  The macromolecules of the present invention, especially dendrimers, can be particularly useful in facilitating passive targeting of drugs to solid tumors and inflammatory sites. This targeting is possible because the vasculature associated with tumors and inflammation has increased permeability to macromolecules and limited lymph drainage.

  The macromolecules of the invention can also be targeted to specific cell types or tissue types using targeting moieties present on dendrimers.

Thus, in yet another aspect of the invention, having controlled end group stoichiometry, and
A first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a targeting moiety for targeting the pharmaceutically active agent and / or macromolecule to one or more specific cell or tissue types A dendrimer is provided, wherein the end group stoichiometry refers to the number and type of end groups.

  Examples of suitable targeting moieties include lectins, antibodies and functional fragments of antibodies. The targeting moiety may also include a cell surface receptor ligand.

  Several different cell surface receptors serve as targets for macromolecular binding and / or uptake. In particular, receptors useful in the present invention and related ligands include folate receptors, adrenergic receptors, growth hormone receptors, luteinizing hormone receptors, estrogen receptors, epidermal growth factor receptors, fibroblasts These include, but are not limited to, growth factor receptors (eg FGR2), IL-2 receptor, CFTR and vascular epidermal growth factor receptor.

In yet another embodiment of the present invention, a polymer with controlled end group stoichiometry, comprising a surface layer, at least one subsurface layer, and the remainder of the pharmaceutically active agent, derivative or precursor thereof. A macromolecule comprising at least two end groups, including a first end group that is a group and a second end group capable of functioning as a cell receptor ligand (in this case, end group stoichiometry is defined as Refers to number and type).

In yet another embodiment of the present invention, a polymer with controlled end group stoichiometry, comprising a surface layer, at least one subsurface layer, and the remainder of the pharmaceutically active agent, derivative or precursor thereof. A macromolecule comprising at least two end groups including a first end group that is a group and a second end group capable of functioning as a cell receptor (in this case, end group stoichiometry is the number of end groups) And type).

  Folic acid is an essential vitamin for nucleotide base biosynthesis and is therefore required in large quantities in proliferating cells. In cancer cells, this increased folate requirement is often reflected in the overexpression of folate receptors responsible for transport of folate across the cell membrane. In contrast, folate uptake into normal cells is promoted by reduced folate transporters rather than folate receptors. Folate receptors are upregulated in many human cancers, including ovarian, brain, kidney, breast, myeloid cells and lung malignancies, and the density of folate receptors on the cell surface increases as cancer develops It seems to increase.

  The relative specificity of folate receptors for tumor cells, particularly advanced tumor cells, means that folate, a folate receptor ligand, can be a useful candidate for targeting chemotherapeutic drugs to tumors. The specificity of the interaction between the folate receptor and the folate receptor ligand-chemotherapeutic conjugate is determined by the cell surface expression pattern of the folate receptor found between certain nontransformed and malignant transformed epithelial cells. The difference is further strengthened. In the case of non-transformed cells, folate receptors are preferentially expressed on the apical membrane of cells facing the body cavity and inaccessible to the reagents present in the blood. However, when transformation occurs, the cell loses its polarity and the receptor can gain access to drugs in the circulatory system targeted to the folate receptor. Thus, folic acid or folic acid derivatives can be useful targeting moieties for the polymers of the present invention.

In yet another embodiment of the present invention, a polymer with controlled end group stoichiometry, comprising:
A polymer having two or more different end groups, including a first terminal group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a second terminal group that is the residue of folic acid or a folic acid analog (In this case, end group stoichiometry refers to the number and type of end groups).

  In a preferred embodiment of this aspect of the invention, the pharmaceutically active agent is an antitumor pharmaceutical agent. Cytotoxic agents, cytokines, anti-angiogenic agents, anti-mitotic agents, etc. can be used, or any combination thereof can be used.

  In a preferred embodiment, the pharmaceutically active agent is selected from one or more of methotrexate, taxol, cisplatin, carboplatin and doxorubicin.

  In the synthesis of the dendrimers of the present invention, a linker moiety can be incorporated, for example by use in place of a generation building unit or to mediate the attachment of end groups to the generation building unit.

Accordingly, in a further aspect of the invention, a polymer (preferably a dendrimer) with controlled end group stoichiometry, comprising:
One or more linker moieties,
Two containing a first terminal group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a second terminal group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule A polymer having these different end groups, and the first and / or second end groups are attached to the polymer framework by a linker moiety (in this case, end group stoichiometry is the number and type of end groups Pointing).

  In yet another embodiment, the second end group is a moiety that facilitates targeting of the pharmaceutically active agent and / or macromolecule to one or more cell types or tissue types and one or more cell types or tissue types. Selected from moieties that facilitate incorporation of the pharmaceutically active agent and / or polymer into the one or more linker moieties comprising PEG.

  The linker may be cleavable or non-cleavable depending on the requirements of the group attached to the surface. A cleavable linker can be designed to be cleaved enzymatically, for example, for dendrimers targeted to tissues that express those enzymes. Alternatively, an acid labile linker may be preferred so that the compound attached thereto is released under acidic conditions such as hypoxic tissue.

Overview of various linkers The linker portion can be selected from one or more of the following.

Amide linker (19-21)
The nature of the amide bond is important in determining whether free drug is released from the conjugate. For example, conjugating a drug (ie, doxorubicin) to a carrier through an amide bond produces a conjugate that is stable to hydrolysis and does not exert any anticancer activity in vitro. Also, a drug directly bonded to a carrier via an amide bond is not easily cleaved as a free drug, but rather cleaved as a drug-amino acid when the carrier itself is degradable. Release of free drug from a carrier directly linked through an amide linker is achieved only in rare circumstances where the drug itself is a peptide-like molecule and the bond between the drug and the carrier is enzymatically cleavable. You won't be able to do it.

Hydrazones, oximes and imine linkers (21-25)
Hydrazone, oxime and imine linkages do not require the presence of an enzyme to cleave the drug from the carrier. These can be hydrolytically cleaved at C = N bonds in low pH environments such as in tumor extravascular space or in lysosomes. Commonly used hydrazones, oximes, and imine linkers result from the reaction of the hydrazine, alkoxyamine, or amine moiety of the linker with the carbonyl (ketone or aldehyde) of the pharmaceutically active moiety, respectively. This linkage is replaced by modifying the number of alkyl groups surrounding the C = N bond moiety, or with an electron withdrawing moiety (to increase acid instability) or an electron donating moiety (to reduce acid instability). In some cases, modification may be performed so that the rate of hydrolysis is reduced.

Ester linker (19, 26)
Acid labile and metabolizable ester linkers can be made. Orthoesters have been used to conjugate PEG to lipids that bind anionic membrane carriers. The stability of this conjugate in acidic conditions (pH 4-6) depends on the structure of the ester or orthoester linker. In general, α-methoxy-ω- {N- (2-octadecyloxy- [1,3] dioxolan-4-yl) methylamide} -polyethylene glycol ₁₁₀ exhibits good stability at both pH 4 and pH 5, and α-methoxy- ω- {N- (2-cholesteryloxy- [1,3] dioxolan-4-yl) methylamide} -polyethylene glycol ₁₁₀ is very stable at pH 5, but slightly unstable at pH 4, and α-methoxy -ω- {N- (2-methyl-s-octadecyloxy- [1,3] dioxan-5-yl) -amide} -polyethylene glycol ₁₁₀ and α-methoxy-ω- {N-2- (3-hydroxy Propyl-cholesterylcarbamate) -2-methyl- [1,3] dioxane-5-yl-amide} -polyethylene glycol ₁₁₀ is not stable. For simple ester conjugation to small molecules, the diester functionality provides more metabolic cleavage sites compared to monoesters that are more stable than disulfides but less stable than amide bonds.

Peptide linker (27-33)
Peptide linkers are by far the most versatile of all cleavable linkers because many different combinations of amino acids can be used to control the cleavage rate and cleavage enzyme. However, in using these linkers as drug-carrier conjugates, these linkers are generally 1) they are cleavable by non-specific peptidases in the body and are therefore non-specific at sites of non-tumor distribution. There are two problems: it can lead to significant drug toxicity and 2) cleavage can occur at sites in the linker where amino acids remain attached to the drug molecule. This can interfere with the chemotherapeutic action of drug molecules. Alternatively, the bound amino acid does not change the pharmacological action of the drug, but may affect its pharmacokinetics. However, these cleavage effects can be controlled by appropriate selection of the amino acid (eg, proline) in the peptide linker that is directly attached to the drug molecule.

  In general, linkers capable of cathepsin B cleavage have been designed to be cleaved by the lysosomal system after drug conjugate endocytosis. This is because cathepsin is located in the lysosome and is not released in the cytosol. Endocytosis is generally initiated following the binding of a carrier (which is usually an antibody to a cancer-specific cell surface receptor or a ligand for a cancer-specific cell surface receptor) to the cell membrane.

  Nonspecific proteases (ie, proteases that are not specific for a particular peptide sequence) can cleave drugs from PEGylated dendrimers after they have undergone sufficient extravasation and accumulation in tumor tissue .

  The following guidelines apply for the rate of peptide cleavage (where a> b indicates that the cleavage rate of a is greater than the cleavage rate of b): For peptide sequences used as a linker between the active drug and the dendrimer terminal nitrogen, terminal CG> no terminal CG> terminal G = terminal GFG> terminal GGG and terminal GGGF = terminal GPG. Note: CG bonds are reduced by GSH. GGG bonds are generally very stable compared to other peptide bonds. Dipeptide cleavage is generally specific for a particular protease and can be controlled based on the expression of various proteases contained within the tumor cells.

Glutaraldehyde (34, 35)
Glutaraldehyde is generally not used as a normal linker, but is used as a stabilizer, particularly in gel formulations, or used to covalently attach a drug to a desired adsorption surface. It is also used as a non-metabolizable spacer and creates a gap between the drug molecule and the large carrier by a crosslinking reaction.

PEG-peptide (2, 36)
PEG-peptides are used in the same way as normal peptides except that the PEG moiety adds in vivo stability and mass to the carrier. Typically used to conjugate a drug to an antibody carrier, increasing the distance between the Ab and the drug while simultaneously exposing the enzymatic cleavage site, reducing the immunogenicity of the conjugate, and It has the advantage of increasing the circulation time and increasing the solubility of the complex. For adriamycin and duocarmycin derivatives, antiproliferative effects have been observed after conjugation internalization and enzymatic release of the active drug, which is not necessarily released as a free drug.

Disulfide linker (1, 37, 38)
Disulfide linkers are the most labile of currently used linkers and undergo rapid reductive cleavage in vitro. However, its in vivo stability is generally higher than its in vitro stability. These can be formed by disulfide bonds between sulfur-containing amino acids or can be formed by non-peptide based disulfide bonds. They are highly reactive with other nucleophilic thiols in the body and thus show rapid plasma clearance.

General overview of linker cleavability In circulation, the order of linker cleavability is as follows:
Disulfide> long peptide ≧ ester> hydrazone ≧ tetrapeptide (GGGF) = tripeptide (GFG> GGG = GPG) ≈or> dipeptide (AV, AP, GP, FL, V-Cit)> glutaraldehyde = amide.

Linker Recommendations The stability of various linkers depends on the group to which they are conjugated (ie the accessibility of the enzyme to the linker), the behavior of the conjugate at the site where activity is required (ie cellular uptake or extracellular accumulation). As well as the nature of the conjugate (ie ester vs. amide). In current systems, the in vivo behavior of disulfide conjugates is expected to be relatively difficult to predict. Long-chain peptides are more readily accessible to proteases and are cleaved rapidly, but the cleaving may be too rapid and may occur at non-specific sites and may not be biologically active Release of active peptide / amino acid species may occur.

  Cleavage of the C = N based linker (hydrazone, oxime or imine), ester or peptide conjugate occurs over at least several days, allowing the conjugate to accumulate in tumor tissue. Each has advantages, but an ester or hydrazone linker may be preferred. The ester bond linking the pharmaceutically active substance to the dendrimer results in a bond that is rapidly cleaved, which may not be specific for the target site, but cleavage results in the release of the free pharmaceutically active substance. Hydrazone linkages are more stable than esters in systemic circulation, and hydrolysis at the C = N bond results in conjugates that are cleaved with higher specificity at the tumor site. However, to allow hydrazone formation, it may be necessary to modify the pharmaceutically active molecule by incorporation of a carbonyl moiety or a hydrazine moiety.

  In a preferred embodiment, the linker moiety may comprise two reactive functional groups F and Y connected by one or more carbon or heteroatoms (preferably a hydrocarbon backbone). The functional group F can be activated to react with reactive amine moieties such as on the surface of the dendrimer. Typically, the functional group F is a carboxylate group or residue thereof. The other functional group Y is an amine containing a protecting group or is selected to have a specific reactivity complementary to the reactive group of the desired organic group attached to the surface of the dendritic motif. Typical examples of Y include amine, hydroxyl, thiol, alkenyl or alkynyl, nitrile, halide, carboxylate or azide groups.

  When a linker moiety is used to connect the end group to the surface amine group of the dendrimer, the reaction between the linker and the organic group may be performed before reacting the linker moiety with the surface amine of the dendritic motif, This may be done after reacting the linker moiety with the surface amine of the dendritic motif. The reaction used to introduce one or more linker moieties into the dendritic motif is performed to ensure that all of the deprotected surface amines of the dendrimer are fully reacted with the linker moiety. Typically this is done by using an excess of the selected linker moiety.

  In the present invention, a photocleavable linker can be used in addition to the above-mentioned linker. For example, a heterobifunctional photocleavable linker can be used. The heterobifunctional photocleavable linker can be water soluble or organic solvent soluble. These contain activated esters that can react with amines or alcohols and epoxides that can react with thiol groups. Between the ester group and the epoxy group is a 3,4-dimethoxy-6-nitrophenyl photoisomerization group which, when exposed to near-ultraviolet light (365 nm), fully forms the amine or alcohol. discharge. Thus, a pharmaceutically active ingredient linked to a dendrimer using such a linker can be released in a biologically active or activatable form by exposing the target area to near ultraviolet light.

  For example, the alcohol group of taxol can be reacted with an activated ester of an organic solvent soluble linker. This product is then reacted with a partially thiolated dendrimer surface. In the case of cisplatin, the amino group of the drug can be reacted with a water-soluble linker. If the amino group does not have the required activity, it can be reacted with a primary amino-containing active analog of cisplatin, such as Pt (II) sulfadiazine dichloride. When conjugated in this way, the drug is inactive and will not harm normal cells. When the conjugate is localized within the tumor cell, it is exposed to laser light of the appropriate near UV wavelength to release the active agent into the cell.

In yet another embodiment of the invention, a polymer with controlled end group stoichiometry, comprising a surface layer, at least one subsurface layer, and the remainder of the pharmaceutically active agent, derivative or precursor thereof. A first end group that is a group, and a polymer having two or more different end groups, including a pharmaceutically active agent and / or a second end group selected to modify the pharmacokinetics of the polymer;
Pharmaceutical compositions comprising a pharmaceutically acceptable carrier, diluent or excipient therefor are provided.

  The polymeric component can be a dendrimer, preferably a lysine dendrimer.

  The pharmaceutically active agent can be selected from one or more of the categories of pharmaceutically active agents described above. Preferably, the pharmaceutically active agent is a cancer therapeutic agent and a cancer-related drug including an antimitotic agent or an antimetabolite, an obesity therapeutic agent, an anti-inflammatory agent, or an immunosuppressive agent.

  The second end group can be selected to increase the plasma half-life of the pharmaceutically active substance. In yet another embodiment, the second end group is selected to facilitate targeting and / or uptake of the pharmaceutically active agent to one or more specific cell types or tissue types.

  The second end group can include a polyethylene glycol (PEG) or polyethyloxazoline (eg, PEOX) motif.

In yet another embodiment of the present invention, a polymer with controlled end group stoichiometry, comprising a surface layer, at least one subsurface layer, and the remainder of the pharmaceutically active agent, derivative or precursor thereof. A polymer having two or more different terminal groups, including a first terminal group that is a group, and a second terminal group that is a residue of folic acid or a folic acid analog;
A pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier, diluent or excipient therefor (where end group stoichiometry refers to the number and type of end groups).

  In a preferred embodiment of this aspect of the invention, the pharmaceutically active agent is an antitumor pharmaceutical agent. Cytotoxic agents, cytokines, anti-angiogenic agents, anti-mitotic agents, etc. can be used, or any combination thereof can be used.

  The anti-tumor pharmaceutical agent can be selected from one or more of the following: rituximab, oxaliplatin, docetaxel, gemcitabine, trastuzumab, irinotecan, paclitaxel, bevacizumab, carboplatin, cetuximab, doxorubicin, pemetrexed, epirubicin, bortotezine , Vinorelbine, mitoxantrone, fludarabine, doxorubicin, alemtuzumab, carmustine, ifosfamide, idarubicin, mitomycin, fluorouracil, cisplatin, methotrexate, melphalan, arsenic, denileukin diftitox, cyclotharibin calcium, cytarifamide Etoposide, mistletoe (viscum album), mesna, gemtuzumab, ozoga Sewing machine, busulfan, pentostatin, cladribine, bleomycin, daunorubicin, bendamustine, dacarbazine, raltitrexed, vincristine, hotemustine, etoposide phosphate, porfimer sodium and vinblastine.

  In a preferred embodiment, the pharmaceutically active agent is selected from one or more of methotrexate, taxol, cisplatin, carboplatin and doxorubicin.

  The pharmaceutically acceptable carrier or excipient can be selected from any known carrier or excipient depending on the delivery route selected for the active agent.

  The pharmaceutical composition can be formulated for oral delivery, injection delivery, rectal delivery, parenteral delivery, subcutaneous delivery, intravenous delivery, intramuscular delivery or other delivery. The pharmaceutical compositions can be formulated as tablets, capsules, caplets, ampoules for injection, or ready-to-use solutions, lyophilized substances, suppositories, boluses, or implants.

  The formulation of such compositions is well known to those skilled in the art. Any and all conventional solvents, dispersion media, media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents as suitable pharmaceutically acceptable carriers and / or excipients Etc. The use of such media and agents for pharmaceutically active substances is well known in the art and is described, for example, in "Remington's Pharmaceutical Sciences" (18th Edition, Mack Publishing Company, Bensylvania, USA). Yes. Unless the usual media or agents are compatible with the end groups of the dendrimer polymers described herein, it is contemplated to use them in the pharmaceutical compositions of the present invention. Supplementary active ingredients can also be incorporated into the compositions.

  It is especially beneficial to formulate the composition in dosage unit form for ease of administration and uniformity of dosage. As used herein, a “unit dosage form” is a physically discrete unit suitable as a unit dosage for a human subject to be treated, wherein each unit is a necessary pharmaceutical carrier and / or pharmaceutical. It refers to those containing a predetermined amount of active ingredient calculated to produce a desired therapeutic effect together with a diluent. The specifications for the novel unit dosage forms of the present invention are: (a) the unique characteristics of the active ingredient and the specific therapeutic action to be achieved, and (b) the technology of formulating such active compounds for specific treatments. Are determined by the limitations inherent in and depend directly on them.

  In yet another aspect of the present invention, an effective amount of the macromolecule in the prophylactic or therapeutic treatment of a human or non-human animal patient or in the manufacture of a medicament for treating a human or non-human animal patient. Use is provided.

  In yet another aspect of the invention, a method for treating a disease indicator or physiological defect in a mammalian (including human) patient, wherein a patient in need of such treatment has a prophylactically effective amount or There is provided a method comprising administering a therapeutically effective amount of the pharmaceutical composition.

  A variety of administration routes are available. The particular mode chosen will, of course, depend on the particular condition being treated and the dosage required to achieve therapeutic efficacy. The methods of the invention generally use any medically acceptable mode of administration (ie, any mode that provides a therapeutic level of the active ingredient without causing clinically unacceptable adverse effects). Can be implemented. Such modes of administration include oral, rectal, topical, nasal, inhalation, transdermal or parenteral (eg, subcutaneous, intramuscular and intravenous) routes, intraocular and intravitreal (eye). Route to the vitreous). Examples of the preparation for oral administration include discontinuous units such as capsules, tablets, and oral preparations. Other routes include direct intrathecal administration to the cerebrospinal fluid, direct introduction by various catheters and balloon angioplasty devices well known to those skilled in the art, and intraparenchymal injection into the target area.

  The composition is conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical arts. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, fine solid carriers or both and then, if necessary, shaping the product.

  Compositions of the present invention suitable for oral administration may be presented as discrete units, such as capsules, sachets, tablets or mouthpieces each containing a predetermined amount of a polymer, placed in liposomes, or aqueous Alternatively, it can be presented as a suspension in a non-aqueous liquid, such as a syrup, elixir or emulsion.

  Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active ingredient, preferably one that is isotonic with the recipient's blood. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in polyethylene glycol. Among the acceptable vehicles and solvents that can be employed are water, and isotonic sodium chloride solution. Sterile fixed oils are also conveniently used as solvents or dispersion media. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid are also useful in the manufacture of injections.

  The polymers of the present invention can also be formulated for delivery as a system designed to administer dendrimer polymers by intranasal administration or inhalation, such as a fine aerosol spray containing the active ingredient.

  Another delivery system can be a slow broadcast delivery system. A preferred sustained broadcast delivery system is a system that can cope with the release of the polymer of the present invention in sustained release pellets or sustained release capsules. Various types of Xu Broadcasting system can be used. These include, but are not limited to, (a) an erosion system in which the active ingredient is contained in the matrix, and (b) a diffusion system in which the active ingredient penetrates through the polymer at a controlled rate. Pump-type hardware delivery systems can also be used, some of which are suitable for implantation.

  The polymers of the present invention are administered in a prophylactically effective amount or a therapeutically effective amount. A prophylactically effective amount or therapeutically effective amount is an amount necessary to at least partially achieve a desired effect, or delay the onset of, suppress the progression of, or prevent the occurrence or progression of a particular condition being treated. It means the amount necessary to stop completely. Such amounts will of course depend on the particular condition being treated, the severity of the condition, and individual patient parameters such as age, physical condition, size, weight and combination treatment. These factors are well known to those skilled in the art and can be addressed by routine experimentation alone. In general, it is preferred to use the maximum amount (ie, the highest safe amount according to reasonable medical judgment). However, those skilled in the art will appreciate that lower doses or tolerated doses may be administered for medical reasons, psychological reasons, or virtually any other reason.

  In general, the daily dose of polymer can be from about 0.01 mg / kg / day to 1000 mg / kg / day. Small doses (0.01-1 mg) can be administered initially, and then doses can be increased up to about 1000 mg / kg / day. If the response in the subject is inadequate at those doses, higher doses (or higher effective doses by more localized different delivery routes) may be used to the extent patient tolerance allows. Multiple doses per day may be considered to achieve an appropriate systemic level of compound.

  In yet another preferred embodiment, the pharmaceutically acceptable carrier or excipient is a sterile aqueous salt solution, suspension and solution comprising saline and buffered medium, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's solution. One or more of the emulsions can be selected. Intravenous vehicles include water and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose. For administration by non-intravenous routes, the carrier can take the form of coagulated plasma, preferably the patient's coagulated plasma. Alternatively, the carrier can be a physiologically compatible biodegradable solid or semi-solid that does not contain plasma, such as a gel, suspension or water-soluble jelly. Gum arabic, methylcellulose and other cellulose derivatives, sodium alginate and tragacanth suspensions or gels such as sodium carboxymethylcellulose 2.5%, tragacanth 1.25% and guar gum 0.5% are suitable for use as carriers in the practice of the present invention. .

  In yet another preferred embodiment, the macromolecule in the pharmaceutical composition can comprise at least two different end groups that are pharmaceutically active.

  Such embodiments can be utilized for various types of combination therapy.

In yet another preferred embodiment, the pharmaceutical composition comprises
A polymer (preferably a dendrimer) with controlled end group stoichiometry,
At least one cleavable or non-cleavable linker moiety;
A first terminal group that is the residue of a pharmaceutically active agent, derivative or precursor thereof;
A macromolecule comprising at least two end groups comprising a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule;
A pharmaceutically acceptable carrier, diluent or excipient therefor, wherein the first and / or second end groups are attached to the polymeric framework by one or more linker moieties (this In some cases, end group stoichiometry refers to the number and type of end groups).

  After cancerous tumors grow rapidly and uncontrollably to about 2 mm in diameter, the lack of effective nutrient supply inside the tumor will limit further cell replication (1). Until angiogenesis begins and provides the tumor with its own blood supply, the tumor can remain at this size for many years. One of the mechanisms by which this can occur is a transcription factor that is up-regulated at sites where oxygen and glucose supply is restricted (eg inside the tumor mass), leading to up-regulation of genes involved in angiogenesis This is due to the activation of hypoxia-inducible factor-1α (2). However, rapid vascularization within the tumor creates a vascular architecture with large gaps between vascular endothelial cells, which allows the accumulation of large particles that are not normally permeable in normal vasculature. There is also a tumor EPR effect because lymph drainage to remove accumulated particles does not occur in the tumor mass (1).

  Anomalous metabolism and vascular supply within the tumor results in a different pH gradient than normal cells. Increased accumulation of lactic acid and carbonic acid within the tumor results in a slightly acidic extracellular environment (pH ~ 6.5), and due to a defect in the Na / H exchanger, the intracellular environment is neutral or slightly alkaline (pH 7. 0 to 7.4). This unique pH gradient prevents the accumulation of some chemotherapeutic drugs that are weak bases, such as doxorubicin. The acidic extracellular environment has also been linked to cancer metastasis by degradation of the stromal matrix and intercellular gap junctions.

  This extracellular acidity can be used to allow release of anticancer drugs by pH from carrier molecules linked by acid labile linkers. Release can occur in the extracellular space (pH about 6.5) or in the lysosomal compartment (pH about 4-5). Anticancer drugs are also linked to tumor targeting groups, such as transferrin, in which most tumor cells overexpress their receptors. Other linkers that can be cleaved by cathepsins B or D that are overexpressed in tumor lysosomes have also been used.

Accordingly, in yet another aspect of the invention, a method for treating a tumor (including a malignant tumor) in a mammalian (including human) patient in need of treatment of the tumor (including a malignant tumor), To the patient,
A polymer with controlled end group stoichiometry, comprising a surface layer, at least one sub-surface layer, and a first end group that is the residue of an antitumor pharmaceutical agent, derivative or precursor thereof,
A macromolecule comprising at least two end groups comprising an anti-tumor pharmaceutical agent and / or a second end group selected to modify the pharmacokinetics of the macromolecule;
A method comprising administering an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient therefor (where end group stoichiometry is the number and type of end groups Is provided).

  In certain embodiments, the polymer further comprises one or more linker moieties, wherein the one or more linker moieties attach the first and / or second end groups to the polymer framework.

  The anti-tumor pharmaceutical agent can be of any suitable type. Cytotoxic agents, cytokines, anti-angiogenic agents, anti-mitotic agents, etc. can be used, or any combination thereof can be used.

  The antineoplastic agent can be selected from one or more of the following: rituximab, oxaliplatin, docetaxel, gemcitabine, trastuzumab, irinotecan, paclitaxel, bevacizumab, carboplatin, cetuximab, doxorubicin, pemetrexed, epirubicin, bortepomibine , Mitoxantrone, fludarabine, doxorubicin, alemtuzumab, carmustine, ifosfamide, idarubicin, mitomycin, fluorouracil, cisplatin, methotrexate, melphalan, arsenic, denileukin diftitox, denitalukin diftitox, cytarabine, levoforifamide Mistletoe (viscum album), mesna, gemtuzumab, ozogamicin , Busulfan, pentostatin, cladribine, bleomycin, daunorubicin, bendamustine, dacarbazine, raltitrexed, vincristine, hotemustine, etoposide phosphate, porfimer sodium and vinblastine.

  In a preferred embodiment, the anti-tumor pharmaceutical agent is selected from one or more of methotrexate, taxol, cisplatin, carboplatin and doxorubicin.

  The second end group can be selected to increase the plasma half-life of the pharmaceutically active substance. The second end group can be selected to facilitate targeting and / or uptake of the anti-tumor pharmaceutical agent to one or more specific cell types or tissue types. In a preferred embodiment, the second end group comprises polyethylene glycol (PEG group) or polyethyloxazoline.

  The pharmaceutically acceptable carrier or excipient can be selected from any known carrier or known excipient, depending on the delivery route selected for the active agent.

  The polymers of the invention, in particular dendrimers, can be used to target pharmaceutically active agents to the lymphatic system.

  The lymphatic system consists of a complex network of specialized vessels, nodes and areas of lymphatic tissue that are distributed throughout the vascular region of the body. Lymphatic vessels are primarily responsible for maintaining fluid balance, but also play a role in maintaining intestinal absorption and transport of neutral fat and effective immune defense mechanisms. In most capillary beds, the vascular endothelium is continuous, with an unbroken basement membrane. Capillaries are therefore relatively poorly permeable to large and small particles injected into the interstitial space (eg, by subcutaneous or intramuscular injection). In contrast, capillary lymph vessels consist of a monolayer of overlapping endothelial cells with an incomplete basement membrane. This results in an endothelial layer with an “opener” intercellular junction than that found in capillaries. Estimates of intercellular junction distances range from a few microns (3-5) to 15-20 nm (5-10). Thus, these large intercellular junctions can facilitate preferential transport or excretion of macromolecules, colloids and potentially dendrimers from the interstitial space to the lymphatic vessels.

  In terms of directed or targeted delivery to lymphatic vessels, lymph also serves as the primary conduit for tumor metastasis dissemination and is widely used as a target for cytotoxic agents designed to counter the spread of metastases from solid tumors. It's being used. B lymphocytes and T lymphocytes, which are present in relatively high concentrations in the lymph, are also attractive targets for cytokines such as interferons and immunomodulators in general. In addition, recently, in human immunodeficiency virus (HIV) positive patients, lymphoid tissue has been found to have increased viral load and increased viral growth, which has led to interest in lymph as a therapeutic target in the treatment of HIV and AIDS. Is growing. To date, several liposomal formulations that provide targeted delivery of drugs to lymph nodes have been developed, particularly with respect to the anticancer drug doxilubicin (Doxil / Caelyx) (16-21).

Accordingly, in yet another aspect of the invention, a method for targeted delivery of a pharmaceutically active agent to an animal's lymphatic system, comprising:
A polymer with controlled end group stoichiometry, a surface layer, at least one subsurface layer, and a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof,
An effective amount of a macromolecule comprising at least two end groups comprising a second end group selected to facilitate uptake of the pharmaceutically active agent and / or macromolecule into the lymph;
• A method is provided comprising administering a pharmaceutically acceptable carrier, diluent or excipient therefor.

  In a preferred embodiment, the second end group is PEG or polyethyloxazoline. In a preferred embodiment, the PEG groups are relatively monodisperse and are selected from a molecular weight range of 200 to 10,000 daltons, more preferably the PEG groups are selected from a molecular weight range of 500 to 5,000 daltons. In yet another preferred embodiment, the second end group is PEG with a molecular weight greater than about 1000 Daltons. In yet another preferred embodiment, the second end group is a PEG motif having a molecular weight greater than about 1500 daltons.

  In a preferred embodiment, the dendrimer has a molecular weight greater than about 20 kDa, preferably greater than about 30 kDa, more preferably greater than about 50 kDa.

  In a preferred embodiment, the dendrimer comprises a cleavable or non-cleavable linker moiety that attaches the first end group to the dendrimer skeleton.

  In a preferred embodiment, dendrimers are administered by subcutaneous injection to animals, including humans.

In yet another embodiment, the present invention provides a polymer having controlled end group stoichiometry having a surface layer, at least one subsurface layer, and two or more different end groups. At least one end group is a pharmaceutically active agent, derivative thereof, precursor thereof, or residue thereof, and at least one end group is selected to modify the pharmacokinetics of the pharmaceutically active agent (this In some cases, end group stoichiometry refers to the number and type of end groups).

  The process for synthesizing the dendrimers of the present invention involves the sequential reaction of a growing dendrimer core moiety with one or more layers of lysine or lysine analogs as generation building compounds. The apical carboxylate F of the lysine analog, which represents the unique point where the dendritic motif is attached to the growing polymer core during the synthesis process, is inevitably required prior to reaction with the unprotected amine moiety. Will be activated. Each of the amine groups A and B of the lysine analog is protected to prevent self-condensation. When the carboxylate F of the generation building compound is reacted with the unprotected nitrogen of the growing dendrimer, the amines A and B of the generation building compound are always protected. Furthermore, the reaction of the unprotected amine with the activated lysine analog is always carried out in a manner that ensures that the unprotected amine reacts completely with the selected lysine analog. . This is most simply done by using a stoichiometric excess of activated lysine analog.

  The process for synthesizing the dendrimers of the present invention may involve the reaction of the unprotected amine of the growing dendrimer with a pharmaceutically active agent, a cell surface ligand, and a linker or end group such as PEG. In either case, the linker or terminal carboxylate group is activated for amide bond formation prior to the reaction or in situ. The amine of the linker group is protected or already reacted with the end group. Furthermore, the reaction of the unprotected amine of the growing dendrimer with an activated linker or end group typically results in the unprotected amine being removed by using an excess of the activated group. It is carried out in such a way that it is guaranteed to react completely with the activated group.

  The order in which protecting groups are removed determines the sequence of reactions that can be used to produce dendrimers containing different amine protecting groups, and in particular the cleavage conditions for certain amine protecting groups can lead to the loss of bystander amine protecting groups. In some cases, it can be an important factor. The following table of protecting groups shows a preferred set of resolvable amino protecting groups and ortho amine protecting groups.

  A set of resolvable amine protecting groups is defined as a set in which there is an order of removal such that groups that are not intended to cleave are inert to the cleavage conditions. When a protecting group is defined as orthogonal, this means that each group is inert to the cleavage conditions required to remove each of the other groups in the orthogonal set. Information on exemplary amine protecting groups can be found in the following references: Greene, TW and Wuts, PGM “Protective groups in Organic Synthesis” (3rd edition, John Wiley and Sons, New York, 1999), Kocienski, PJ "Protecting Groups" (3rd edition, Thieme, Stuttgart, 2004). Preferred amine protecting groups can be selected from Table 2.

注：
1．表の第1列に挙げた保護基と、表の最上段に挙げた保護基との組み合わせを、「分割可能」（R）または「オルトゴナル」（O）と定義する。ある組み合わせが「分割可能」とみなされる場合、カッコ内の保護基は、最初に除去されるべき基を表す。
2．2-クロロ-Cbzおよび2-ブロモ-Cbzを指す。
3．分割可能でもオルトゴナルでもない組み合わせ。

Note :
1． The combination of the protecting group listed in the first column of the table and the protecting group listed at the top of the table is defined as “divisible” (R) or “orthogonal” (O). When a combination is considered “dividable”, the protecting group in parentheses represents the group to be removed first.
2. Refers to 2-chloro-Cbz and 2-bromo-Cbz.
3． Combinations that are neither split nor orthogonal.

As yet another aspect, the present invention provides:
(I) a growing polymer comprising an outer layer having a functional group and two or more different protecting groups,
A precursor of a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer. Supplying a precursor;
(Ii) deprotecting the functional group on the outer layer by removing the first protecting group;
(Iii) activating one of the first or second end group precursors;
(Iv) reacting the deprotected functional group with an activated end group precursor;
(V) deprotecting the functional group on the outer layer by removing the second protecting group;
A controlled end comprising: (vi) activating the other first or second end group precursor; and (iv) reacting the deprotected functional group with the activated end group precursor. A process for producing a polymer with group stoichiometry (where end group stoichiometry refers to the number and type of end groups) is provided.

As yet another aspect, the present invention provides:
(I) a growing polymer comprising an outer layer having a functional group and two or more different protecting groups,
A first terminal group precursor that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a second terminal group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer, and Providing a linker moiety comprising a carboxylate group and a protected amine group;
(Ii) deprotecting the functional group on the outer layer by removing the first protecting group;
(Iii) activating a carboxylate group on the linker moiety (iv) reacting the deprotected functional group with an activated carboxylate group on the linker moiety;
(V) deprotecting the amine group on the linker moiety;
(Vi) activating one of the first or second end group precursors;
(Vii) reacting a deprotected amine group with an activated end group precursor;
(Viii) deprotecting the functional group on the outer layer by removing the second protecting group;
Controlled end group chemistry comprising (ix) activating the other first or second end group precursor, and (x) reacting the deprotected functional group with the activated end group Provides a process for producing a polymer with stoichiometry, where end group stoichiometry refers to the number and type of end groups.

A preferred process for synthesizing the polymers of the present invention involves the preliminary step of providing a surface modifier compound for attachment to a growing polymer. The surface modifier compound is
A carboxylate group to facilitate attachment of the modifying compound to a growing polymer,
A first terminal group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and / or a second terminal group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule .

  The surface group stoichiometry (number and type) of the end groups can be controlled by the use of dendritic motifs with established terminal amine protecting group surface group stoichiometry and topology. It has been observed that such an approach can result in a highly pure dendrimer of the invention.

The surface modifier compound can be produced by any suitable method. In certain embodiments, a process for producing a surface modifier compound comprising:
(i) lysine or lysine analog compounds having two or more different amine protecting groups and carboxylate groups,
A precursor of a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer. Supplying a precursor;
(Ii) deprotecting a primary amine on the protected lysine or lysine analog compound;
(Iii) activating the first or second end group precursor;
(Iv) reacting the activated end group precursor with a deprotected amine group;
(V) deprotecting the secondary amine on the protected lysine or lysine analog compound;
(Vi) activating the other first or second end group precursor; and (vii) reacting said other activated end group precursor with a second deprotected amine group to surface A process is provided that includes obtaining a modifier compound.

  The surface modifier compound preferably comprises a lysine or lysine analog backbone. The surface modifier compound can include multiple lysine or lysine analog motifs in its backbone.

  The process for synthesizing the surface modifier compounds of the present invention can include removing one or more terminal amine protecting groups to obtain one or more reactive amine groups. These reactive amine groups are then reacted with a precursor of the first or second end group. The carboxylate portion of the end group precursor will be activated for amide bond formation prior to reaction or in the system. Furthermore, the reaction of the unprotected amine of the lysine or lysine analog backbone with the activated end group ensures that the unprotected amine reacts completely with the activated end group. In such a manner, it can typically be done by using a stoichiometric excess of the activated group.

  In certain embodiments, the process for producing the surface modifier compound optionally protects the selected carboxylate group prior to removal of the protecting group on the terminal amine present on the lysine or lysine analog backbone. Can be included. The protecting group used for the protected carboxylate group is preferably stable to the conditions required to remove the protecting group present on the terminal amine. Carboxylate protecting groups such as methyl esters, more preferably ethyl esters are suitable. Information on exemplary carboxylate protecting groups can be found in the following references: Greene, TW and Wuts, PGM “Protective groups in Organic Synthesis” (3rd edition, John Wiley and Sons, New York, 1999), Kocienski, PJ "Protecting Groups" (3rd edition, Thieme, Stuttgart, 2004).

  If the synthesis of the surface modifier compound requires an additional deprotection step after the addition of the end group, it is important to consider the stability of this end group for subsequent reactions. In a preferred sequence of end group addition, a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule is first added to the lysine or lysine analog backbone. Furthermore, in situations where a pharmaceutically active agent should be attached to a lysine or lysine analog backbone via a labile linker, the dendritic motif core is preceded by reaction of the unprotected terminal amine group of the lysine or lysine analog backbone. It may be necessary to deprotect the selected carboxylate of the lysine or lysine analog backbone when the selected carboxylate of the lysine or lysine analog backbone is not protected. May need to be activated before the unprotected terminal amine group occurs.

  The process for synthesizing the surface modifier compound can then include further removal of the terminal amine protecting group and reaction with additional end groups to complete the surface modification of the surface modifier compound. If the selected carboxylate of the surface modifier compound has a protecting group, the protected carboxylate may be deprotected after all of the end groups have been attached, or deprotected prior to attachment of the labile linker. Let's go. After the selected carboxylate of the surface modifier compound is deprotected, all subsequent reactions to form an amide bond between the unprotected terminal amine group and the terminal carboxylate are all carboxy groups of the terminal group. The rate will need to be activated prior to the introduction of the surface modifier compound.

  The process for synthesizing the polymer of the present invention is then continued by reacting the unprotected amine of the growing polymer with a surface modifier compound. The carboxylate portion of the dendritic motif will be activated for amide bond formation prior to reaction or in the system. In a preferred method, the carboxylate moiety of the surface modifier compound is activated in the system. This method is preferred, and in the presence of an unmasked hydroxyl moiety on the growing polymer core or surface modifier compound, accidental introduction to the polymer by including water or other hydroxyl donors. It is possible to limit the formation of typical ester bonds. In certain embodiments, the surface modifier compound is attached to the growing polymer via a linker moiety.

Thus, in an alternative embodiment of the invention,
(I) a growing polymer comprising an outer layer having a functional group and one or more protecting groups, and a first terminal group that is the residue of a carboxylate group pharmaceutically active agent, derivative or precursor thereof, Providing a surface modifier compound comprising a second terminal group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule;
(Ii) activating a carboxylate group on the surface modifier compound;
(Iii) deprotecting the functional groups on the outer layer of the growing polymer by removing the protecting groups;
(Vii) a polymer with controlled end group stoichiometry comprising reacting a deprotected functional group on the growing polymer with an activated carboxylate group on the surface modifier compound Is provided, wherein end group stoichiometry refers to the number and type of end groups.

  The polymer of the present invention may comprise a unique attachment point for either the first or second end group. By doing so, it is possible to synthesize a polymer having a single first or second terminal group. Preferably, the end group attached to the unique attachment point is a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule. More preferably, the second end group attached to the unique attachment point is selected to facilitate targeting and / or uptake of the pharmaceutically active agent to one or more cell types or tissue types.

  In an alternative embodiment, the polymer of the present invention may include a single selected attachment point for either the first or second end group.

  For selective mono-protection of polyamine molecules, the general methods are described in the art. Such a method is described in Krapcho and Kuell, Synthetic Commun. 1990 20 2559. In a preferred method, a dendrimer with a unique attachment point is produced from a divalent or trivalent core, in which case only one of the reactive amine moieties is subjected to other amino protection during the process to build lysine dendrimers. It is protected using a protecting group that is inert or orthogonal to the conditions used to remove the group. Then, by repeating the cycle of lysine condensation and amine deprotection, 1-6 generations (more preferred) where there is a single terminal amine moiety that is distinguished from other terminal amine moieties by its unique amine protecting group. Can build 3-5 generation dendrimers. This unique terminal amine corresponds to a site where a single selected molecule, such as a protein or peptide, or targeting molecule can be attached to the dendrimer at that position.

In a preferred form of this embodiment, the polymer has a controlled end group stoichiometry, comprising a surface layer, at least one subsurface layer, and residues of peptides or proteins, derivatives or precursors thereof. A first end group (which is attached to a single selected attachment site on the macromolecule), and selected to modify the pharmacokinetics of the peptide or protein and / or macromolecule A macromolecule comprising at least two end groups comprising a second end group (where end group stoichiometry refers to the number and type of end groups) is provided.

  In a preferred method, a unique terminal amine moiety protecting group is removed and the terminal amine moiety is removed with a haloacetic acid derivative or maleimide derivative, such as 3-maleimidopropionic acid or 4-maleimidobutyric acid, under conditions where an amide bond is formed. React with. General methods for coupling thiol-containing peptides and proteins to such thiol active groups are described in the Pierce 1989 Handbook and General Catalog and references cited therein.

  The invention is described in more detail below with reference to the accompanying examples. However, the following description is merely an example and should in no way be construed as a limitation on the generality of the invention described above.

The following formula is used for dendrimer nomenclature in the following examples:
Core [last complete layer; building unit] _n- [terminal group] _m [incomplete outer layer; building unit] _p [terminal group] _q
Where
The core is a molecule to which the activated lysine generation building unit is attached and will contain at least one amine moiety to which the first layer of the lysine building unit is added;
N is the number of lysine building units on the outermost complete layer of the polymer and p is the number of lysine building units on the incomplete outer layer of the polymer:
M is the number of end groups on the outermost complete building unit layer, e.g. pharmaceutically active moiety or terminal amine protecting group, and q is the number of end groups on the incomplete outer building unit layer Is
In some cases, terminal groups and / or building units may be added to the core, in which case the core is represented as [terminal group] _r [building unit] _s according to the same principle as above.

  In this nomenclature, given the core and outer layers, the size of the polymer can be fully described. This is because only the lysine building units are used in the construction of these polymer structures, and the valence of the core is known. Furthermore, all the surface amine groups of each polymer layer are replaced with a new lysine. This is because when the layer is added, it completely reacts with lysine.

¹星印は、リジン分岐単位のカルボキシル基にアミドとして結合したアミン基を示す。番号記号は、コアまたはリジン分岐単位のアミンにアミドとして結合したカルボキシル基を示す。

^One star indicates an amine group bonded as an amide to the carboxyl group of the lysine branch unit. The number symbol indicates the carboxyl group attached as an amide to the amine of the core or lysine branch unit.

（実施例１） Additional chemical abbreviations are listed in Table 4.

Example 1

  Examples of pharmaceutically active agents that can be used in the present invention include:

である。がん化学療法に用いられる高用量のメトトレキサートは、骨および消化管粘膜の迅速に分裂する細胞に対して毒性副作用を引き起こしうる。 Methotrexate Methotrexate is an antimetabolite used to treat cancer and autoimmune diseases. This works by inhibiting folate metabolism by competitive and reversible inhibition of dihydrofolate reductase. Chemically, methotrexate is N- [4-[[(2,4-diamino-6-pteridinyl) methyl] methylamino] -benzoyl] -L-glutamic acid. Molecular weight: 454.45 C ₂₀ H ₂₂ N ₈ O ₅

It is. High doses of methotrexate used for cancer chemotherapy can cause toxic side effects on rapidly dividing cells of the bone and gastrointestinal mucosa.

である。 Taxol Taxol (paclitaxel), in particular, the conventional therapies are antimitotic agents used as treatments in women with breast and ovarian cancer did not respond. This works by stabilizing the microtubules and facilitating microtubule assembly so that the cells cannot flexibly use their cytoskeleton. Taxol is a tricyclic diterpene and the structural formula is

It is.

という構造式を持つ。ゼニカルの化学名は(S)-2-ホルミルアミノ-4-メチル-ペンタン酸(S)-1[[(2S,3S)-3-ヘキシル-4-オキソ-2-オキセタニル]メチル]-ドデシルエステルである。分子式はC₂₉H₅₃NO₅である。ゼニカルは495.7の分子量を持つ。鼓腸、便意切迫、脂肪便/油性便および軟便を含むいくつかの望ましくない胃腸副作用がある。 Zenical (also called Xenical, Xenecal, Zencal) is a weight control drug for managing obesity. It exerts its therapeutic activity by forming a covalent bond with the active serine residue site of gastric lipase and pancreatic lipase in the lumen of the stomach and small intestine. As a result, the inactivated enzyme cannot be utilized to hydrolyze dietary fat in the form of triglycerides into absorbable free fatty acids and monoglycerides. Chemically, Xenical is

It has the structural formula The chemical name of xenical is (S) -2-formylamino-4-methyl-pentanoic acid (S) -1 [[(2S, 3S) -3-hexyl-4-oxo-2-oxetanyl] methyl] -dodecyl ester It is. The molecular formula is C ₂₉ H ₅₃ NO ₅ . Xenical has a molecular weight of 495.7. There are a number of undesirable gastrointestinal side effects including flatulence, urgency, fatty / oily stool and loose stool.

Indomethacin (also indometacin) is a nonsteroidal anti-inflammatory drug that is commonly used to reduce fever, pain, stiffness, and swelling. This works by inhibiting the production of prostaglandins, molecules that are known to cause these symptoms.

Cyclosporine cyclosporine is an immunosuppressant widely used for the prevention and treatment of graft-versus-host reactions in transplantation. It has also been tested for the treatment of a wide variety of other diseases where immunological factors may have a pathogenic role.
(Example 2)

Lysine dendrimers were prepared in which about 50% and about 75% of the methotrexate-containing dendrimer end groups were drugs (specifically methotrexate). Methotrexate can be conjugated to the dendrimer by stable binding, and the clearance and biodistribution of this construct is determined.

  It may be desirable to increase the size of individual PEG groups as their relative abundance decreases to maintain the required plasma life span and avoid liver uptake. During plasma exposure, the “non-cleavable” nature of methotrexate preserves the integrity of the construct.

（実施例３） Detailed description of compounds:
BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-MTX] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₁₁₀₀ ] ₁₆ [ε-MTX] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₁₇₁₆ ] ₁₆ [ε-MTX] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₂₈₄₅ ] ₁₆ [ε-MTX] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₃₉₇₄ ] ₁₆ [ε-MTX] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [MTX] ₁₆ [PEG ₅₇₀ ] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [MTX] ₁₆ [PEG ₁₁₀₀ ] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [MTX] ₁₆ [PEG ₁₇₁₆₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [MTX] ₁₆ [PEG ₂₈₄₅ ] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [MTX] ₁₆ [PEG ₃₉₇₄ ] ₁₆
Where MTX represents methotrexate 1 conjugated via a γ-carboxylate group. An important intermediate in this approach is 3 prepared according to the method described in Aust. J. Chem. 2002 55 635-645.

(Example 3)

Taxol-carrying dendrimers Examples of taxol-carrying dendrimers include the following:
BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₁₁₀₀ ] ₁₆ [ε-COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₁₇₁₆ ] ₁₆ [ε-COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₂₈₄₅ ] ₁₆ [ε-COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₁₆ [α-PEG ₃₉₇₄ ] ₁₆ [ε-COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [PEG ₅₇₀ ] ₁₆ [COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [PEG ₁₁₀₀ ] ₁₆ [COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [PEG ₁₇₁₆ ] ₁₆ [COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [PEG ₂₈₄₅ ] ₁₆ [COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [PEG ₃₉₇₄ [COCH ₂ CH ₂ CO-Taxol] ₁₆
BHALys [Lys] ₁₆ [ε, ε-PEG ₅₇₀ ] ₈ [COCH ₂ CH ₂ CO-Taxol] ₂₄
BHALys [Lys] ₁₆ [ε, ε-PEG ₁₁₀₀ ] ₈ [COCH ₂ CH ₂ CO-Taxol] ₂₄
BHALys [Lys] ₁₆ [ε, ε-PEG ₁₇₁₆ ] ₈ [COCH ₂ CH ₂ CO-Taxol] ₂₄
BHALys [Lys] ₁₆ [ε, ε-PEG ₂₈₄₅ ] ₈ [COCH ₂ CH ₂ CO-Taxol] ₂₄
BHALys [Lys] ₁₆ [ε, ε-PEG ₃₉₇₄ ] ₈ [COCH ₂ CH ₂ CO-Taxol] ₂₄

Taxol conjugation can be performed using two different derivatives 2 and 4 (see below). The preparation of 2 is described in J. Med. Chem. 1989 32 788-792.

  The lysine dendrimer can contain about 50% and 75% taxol end groups attached via various “cleavable” linkers.

  All lysine dendrimers produced as fully Boc protected forms were synthesized and purified according to the procedures described in International Patent Application WO 95/34595, the contents of which are all incorporated herein by reference. Removal of the Boc protecting group was performed according to the method described in WO95 / 34595.

If the lysine dendrimer needs to contain tritium, it can have a specific activity in the range of 5 μCi to 15 μCi per mg so that scintillation counting techniques can be used to detect dendrimer material in the biological matrix These materials were prepared by diluting (4,5- ³ H) -L-lysine with non-radioactive L-lysine so that the materials were obtained. This tritiated lysine is then used to produce a p-nitrophenol active ester of di-Boc-lysine, which is incorporated into the outer lysine layer of the target lysine dendrimer using the method described in WO95 / 34595. It is.
(Example 4)

^{BHALys [(3 H) -Lys]} 8 Synthesis of _{[D-Lys] 16 [NH} 2] 32
BHALys [( ³ H) -Lys] ₈ [NH ₂ .TFA] ₁₆ (38 mg, 0.01 mmol) in dry DMF (3 mL) was stirred under nitrogen with D-lysine paranitrophenol ester (181 mg, 0.38 mmol) in DMF (4 mL) and triethylamine (65 μL, 0.46 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours, then poured into acetonitrile (60 mL) and stirred for 6 hours. The resulting fine solid was collected by filtration through a 0.45 μm hydrophilic polypropylene membrane filter and dried under reduced pressure. The product BHALys [( ³ H) -Lys] ₈ [D-Lys] ₁₆ [Boc] ₃₂ was a fine cream solid (40 mg, 56%). BHALys [( ³ H) -Lys] ₈ [D-Lys] ₁₆ [Boc] ₃₂ (34 mg, 0.005 mmol) was suspended in CH ₂ Cl ₂ (1 mL) and cooled to 0 ° C. TFA (398 μL, 2.58 mol) was added dropwise and the reaction was left to stir at 0 ° C. for 10 minutes, then warmed to room temperature and stirred for an additional 3 hours. The solvent was removed under reduced pressure, ether was added to the resulting oil and the solution was triturated to give a white solid. The ether was decanted and the solid was rinsed with ether (3 times). After removing the ether, the resulting solid was dissolved in a minimum amount of water and applied to an Amberlyst (A-26 OH) ion exchange column. Collect 100 mL of water from the column and remove the water by lyophilization to give BHALys [( ³ H) -Lys] ₈ [D-Lys] ₁₆ [NH ₂ ] ₃₂ as a white solid (15 mg, 79% ). LC-MS: (Philic TFA, solvation temperature 300 ° C.) Rf (min) 8.45. ESI (+ ve) m / z = 1386.6 (M / 3), 1040.3 (M / 4), 832.3 (M / 5) 694.0 (M / 6) 594.9 (M / 7).
(Example 5)

Plasma clearance and biodistribution of cationic dendrimers
The material buffer reagent was AR grade. Water was obtained from a MilliQ water purification system (Millipore, Australia). Heparin (10,000 IU / mL) was obtained from Faulding (Australia). Saline for injection was obtained in a 100 mL polyethylene bag from Baxter Healthcare Pty Ltd (NSW, Australia). Tritiated L-lysine (1 mCi / ml) was purchased from MP Biomedicals (Irvine, CA). Non-radiolabeled L-lysine was obtained from Sigma Chemical Co (St. Louis, MO). Starscint scintillation cocktail and Soluene-350 tissue solubilizer were purchased from Packard Biosciences (Meriden, Conn.). Protein standards include blue dextran 2000 (2000 kDa), fatty acid free bovine serum albumin (67 kDa), pepsin (35 kDa), trypsin (23.8 kDa), myoglobin (17.6 kDa), ribonuclease A (13.7 kDa), cytochrome C (12.4 kDa) and These were all obtained from Sigma Chemical Co (St. Louis, MO, USA), including vitamin B12 (1.4 kDa). Elution times for higher molecular weight proteins were obtained by injecting 20 μl of Precision Plus protein standard onto the column (Bio-Rad, Hercules, CA, USA).

³ H-labeled dendrimer was prepared and prepared as a lyophilized powder. Purification before feeding was performed by ultrafiltration and size exclusion chromatography (Sephadex LH20, eluted with water), and free base type uncapped amine-terminated dendrimers were obtained using ion exchange. Purity was confirmed by capillary electrophoresis, NMR and mass spectrometry. The specific activity and molecular weight of the dendrimers are listed in Table 5.

For IV administration, ³ HL-lysine (25 μCi) is diluted with non-radiolabeled lysine in phosphate buffered saline (PBS, pH 7.4) to a final specific activity of 20 μCi / mg did.

  All dendrimers were diluted in PBS and frozen at −20 ° C. until use.

Activity determination and specific activity of scintillation counting dendrimers were determined in triplicate by diluting a stock solution containing a known mass in PBS. A portion was then added to 1 mL Starscint and scintillation counting was performed with a Packard Tri-Carb 2000CA Liquid Scintillation Analyzer (Meriden, Conn.). The average of triplicate measurements was used for all subsequent calculations.

All in vivo animal experiment protocols were approved by the Victorian College of Pharmacy Animal Ethics Committee at Monash University (Parkville, Victoria, Australia).

Intravenous Pharmacokinetic Study Prior to dendrimer administration, the jugular vein of rats (male, Sprague Dawley, 250-350 g) and as described in Ali et al. The carotid artery had a cannula (polyethylene tube, 0.96 × 0.58 mm, Paton Scientific (Victor Harbor, Australia)) under isoflurane anesthesia. These cannulas were flushed with heparinized saline (2 I.U. per ml), sealed, and inserted into a subcutaneous pocket behind the neck during recovery. Rats were allowed to recover overnight prior to dosing. However, no food was given for 12 hours before dosing. After this recovery period, rats were housed in metabolic cages so that urine and feces could be collected separately, and a cannula was attached to the swivel / leash assembly to facilitate drug administration and blood collection. Free access to water was always allowed. L-lysine dendrimer was dissolved in 1 ml of phosphate buffered saline (PBS) and administered through an indwelling jugular cannula at a dose of 5 mg / kg by intravenous infusion over 2 minutes. The cannula was then flushed with 0.25 ml of heparinized saline to ensure that all dosage was infused. Thereafter, blood samples (0.15 ml) were collected from the carotid artery at the following nominal time points: before dosing (-5 minutes), at the end of infusion (t = 0), and 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, 480, 1440 and 1800 minutes. Blood samples were immediately placed in tubes containing 10 I.U. heparin and centrifuged at 3500 × g for 5 minutes. Plasma (50 μl) was then added to 1 ml Starscint scintillation cocktail and vortexed before scintillation counting. The quantification limit (20 dpm) for this plasma assay was confirmed using multiple (n = 5) analysis of spiked plasma samples. Accuracy and accuracy were within ± 20%.

In order to understand the fate of the dendrimer in the distribution studies in vivo biological, it was examined in vivo distribution to a variety of major organs. Once the pharmacokinetic study (30 hours) was completed, the animals were sacrificed by injecting a lethal dose of sodium pentobarbital and the following tissues were removed by dissection: heart, lung, liver, spleen, pancreas, kidney and brain . These tissues were stored frozen (−20 ° C.) in weighted polypropylene tubes until just prior to tissue processing and analysis.

  First, the tissue was processed by homogenizing the sample with 5-10 ml MilliQ water for 10 seconds × 5 times in a Waring mini blender (Extech Equipment Pty. Ltd., Boronia, Australia). In developing a method for measuring relatively low levels of radioactivity in tissue samples, we faced problems associated with chemiluminescent reactions and color quenching that resulted in significant variations in background counts during scintillation counting. These problems also seemed to be exacerbated by increased temperature, exposure to light, and sample agitation. To overcome these problems, the subsequent processing of the organization was performed as a two-step process.

  The first “screening” step was used to determine the approximate level of radioactivity contained in the sample. At this stage, a single sample (typically 40-100 mg of tissue) from each tissue homogenate was placed in a 20 ml polypropylene scintillation vial containing 2 ml of Soluene and tissue digestion was allowed to proceed at 60 ° C. overnight. Isopropanol (2 ml) was then added and the solution was heated at 60 ° C. for an additional 2 hours. After cooling to room temperature, hydrogen peroxide (30% w / v) was added to the sample in 100 μl portions, then it was allowed to stand at room temperature until foaming ceased. Then Starscint (12 ml) was added and the mixture was vortexed before storing the sample at 4 ° C. for 96 hours without stirring in the dark. The sample was then scintillation counted while the counter was maintained at 12 ° C. using a cooled sample tray. Further, in order to minimize the heating during counting, the samples were counted in groups of 6 to 12 samples. Samples were collected from the organs of untreated rats one by one and processed in the same manner to obtain a background count value that could be attributed only to the processing method. These values were subtracted from the values obtained in the screening stage. The data obtained from this screening stage provided a rough indication of the expected amount of radioactivity in each sample. This information was needed for the final analysis experiment.

In the second “analysis” stage of the tissue counting process, tissue samples were analyzed in triplicate. Blank tissue from untreated rats was also processed as described above (but in triplicate) to prepare for background correction. The homogenized tissue of rats dosed with dendrimer is generally in the above screening stage, except that an additional step was taken to compensate for the decrease in radioactivity counting efficiency (quenching) due to the “extraction” process from the tissue. It was processed in the same way as described. To allow correction of counting efficiency, a second set of the same tissue from treated rats was processed in the same way, except that a known amount of radiolabeled dendrimer was first spiked prior to the addition of Soluene. The tissue was spiked with a level of radioactivity approximately equivalent to the radioactivity measured in the screening sample (hence screening data is required). Next, the processing efficiency was calculated as follows:
Efficiency = [(spiked tissue) _DPM- (tissue) _{DPM, uncorrected} ] / (spiked solution) _DPM (1)

(Spiked tissue) _DPM is the mass-corrected radioactivity measured in the spiked sample. (Tissue) _{DPM, uncorrected} is the mass-corrected radioactivity in the tissue sample to which no extra radioactivity spikes are added. The _DPM was a known amount of excess radioactivity added to the spiked sample. In effect, this calculation gives an indication of counting efficiency by using a known (spiked) amount of radioactivity in each tissue as a reference standard.

This efficiency value is then used to correct the ³ H content in the processed sample.
(Organization) _{DPM, Correction} = (Organization) _{DPM, Uncorrected} / Efficiency (2)

  Next, the radioactivity in the whole organ was calculated by knowing the mass fraction of the whole organ present in the processed sample. Results are expressed as a percentage of injected dose in the organ at sacrifice or as a percentage of injected dose per gram of tissue. Due to processing variations, the LOQ may not be easily determined for each organization. Instead, triplicate samples yielding% CV higher than 20% were repeated or classified as below quantifiable levels if no reproducible data was obtained.

Whole blood pharmacokinetics To determine whole blood pharmacokinetics, separate groups of animals were administered the same amount of dendrimer solution, and in a heparinized Eppendorf tube in the same nominal period used for plasma collection. A blood sample (150 μl) was collected. Each blood sample (50 μl) was added in duplicate to two 20 ml scintillation vials. First of all, one vial is not processed, and the other vial (as described above) is studied with a known amount (approximately 300-600 decay per minute (DPM)) to obtain whole blood counting efficiency data. The subject's dendrimer was spiked. The samples were then solubilized overnight at 60 ° C. in 4 ml of a 1: 1 ratio of Soluene-350 and isopropyl alcohol. The sample was then cooled and bleached with 200 μl hydrogen peroxide (30% w / v) before adding Starscint (12 ml). The samples were then left at room temperature for 24 hours before scintillation counting. The counts obtained from the blood samples were corrected for efficiency by comparison with the data obtained for the spiked samples as described above.

Table 6 shows the pharmacokinetic parameters of ³ H-dendrimer in plasma and whole blood after intravenous administration.

Urine was collected from rats receiving urine and fecal dendrimers over three time intervals of 0-8 hours, 8-24 hours and 24-30 hours post-dose. Blank urine samples obtained from each rat prior to dosing were also collected. 100 μl of urine obtained from each time interval was added to 1 ml Starscint and the mixture was vortexed before scintillation counting. After subtracting the background, the radiolabel content of the sample was corrected for the total volume of urine collected during that time interval and converted to a percentage of the total ³ H dose administered.

The stool was collected in a weighed vial and the sample was homogenized into a slurry by immersion in MilliQ water and then dried at 60 ° C. Next, 6 replicate replicate fecal samples (20 mg) were divided into two groups of n = 3 samples. One sample group was processed without further additions, and one group of 3 samples had a known amount of radioactive dendrimer (approximately 500 DPM) to obtain stool count efficiency data (as described above). Spiked. The sample was then solubilized using the method described by Lyons et al. (2000). Briefly, 2 ml of Soluene was added to rehumidified feces and heated at 60 ° C. overnight. Then another 2 ml of isopropyl alcohol was added and the sample was heated for another 2 hours. The solubilized sample was then bleached with 400 μl hydrogen peroxide (30% w / v) before 12 ml Starscint was added. The sample was then allowed to stand at room temperature for 4 hours and then cooled at 4 ° C. for 24 hours, after which scintillation counting was performed at 12 ° C. Using the total dry weight of the collected stool, the total amount of ³ H excreted in the stool was calculated. The LOQ for this assay was estimated to be 3 times the average count in blank feces. This is because the resulting fecal mass fluctuates greatly and therefore the smallest quantifiable count at a given sample mass could not be determined. These results are summarized in Table 7.

Pharmacokinetic calculation The concentration of radiolabel in the plasma / whole blood sample was converted to ng equivalent concentration using the specific activity of the radiolabeled dendrimer. These concentrations are expressed as ng equivalent / ml throughout the report, which assumes that this approach maintains the ³ H radiolabel associated with the complete dendrimer. Note that this may not be the case in some cases, as described below.

The initial decay rate of the plasma concentration-time curve was estimated by linear regression of at least 3 points in the initial phase of the log-linear plasma concentration vs. time plot. Although rate constants obtained from these data are listed as “disappearance” rate constants (K), they do not represent true terminal phase disappearance rate constants. This is because the disappearance was so fast that it was difficult to accurately describe the distribution and disappearance events. The half-life of the initial decay was estimated from t _1/2 = 0.693 / K. In either case, the initial volume of distribution (V _c) during was calculated from the dose / C _p ° (wherein, C _p ° is, t = 0 (i.e. at concentrations in plasma at the end) of infusion over 2 minutes is there). Due to the very rapid initial elimination from plasma and subsequent unusual profiles, further detailed pharmacokinetic assessment of data including confirmation of true elimination rate constant and elimination half-life could not be performed.
(Example 6)

Size Exclusion Chromatography on the biological sample obtained in Example 5 Several methods were used to determine the size of molecular species present in the plasma sample. First, a coarse size separation was performed by eluting plasma samples under gravity through a PD10 gel filtration column (Pharmacia) and collecting fractions. In this method, a plasma sample (500 μl) was diluted with 2 ml of PBS and then placed on a pre-equilibrated PD10 column. Fractions (500 μl) were collected manually into a microfuge tube and 100 μl of them were added to 1 ml Starscint before scintillation counting. The complete dendrimer and lysine solutions in PBS were also eluted through the column to characterize the retention capacity of the pure components.

  To obtain further information regarding the size of the radiolabeled molecular species in plasma, an analytical size exclusion column (Superdex Peptide 10/300 was then coupled to a Waters 590 HPLC pump (Millipore Corporation, Milford, Mass., USA). GL, Amersham Bioscience) for more accurate separation. The plasma sample was again diluted in the same volume of PBS and 100 μl of the mixture was injected onto the column. Samples are eluted with 0.5 ml / min PBS containing 0.3 M NaCl (pH 3.5) and fractionated at 1-minute intervals using a Gilson FC10 fraction collector (John Morris Scientific Pty. Ltd., Victoria, Austria) Collected. A portion was then mixed with Starscint (3 ml) and analyzed by liquid scintillation to determine the radioactivity in each fraction. Separately, the complete dendrimer and lysine in PBS were eluted again through the column to characterize its retention capacity. Dendrimers and lysine were also incubated in fresh heparinized plasma for 1 hour and the dendrimer or lysine-plasma mixture was analyzed by SEC. This analysis was performed in order to obtain a measure of the possibility that a physical interaction between a dendrimer or lysine and plasma components could result in the production of high MW molecular species in the plasma.

To better resolve high MW radiolabeled species present in plasma, samples were also analyzed using a Superdex 75 HR 10/30 size exclusion column. Fractions (0.5 ml) were again collected at 1 minute intervals, diluted in Starscint (3 ml) and analyzed by liquid scintillation counting to determine the radioactivity in each fraction. A standard curve (linear R ² = 0.9915) was generated using the protein standard and was used to estimate the molecular weight of the eluted protein. Protein elution was monitored at 280 nm using a Waters 486 UV detector (Millipore Corporation, Milford, Mass., USA). Column void volume was determined using Blue Dextran 2000.
(Example 7)

Synthesis of tritium-labeled anionic dendrimers
^{_{BHALys [3 H-Lys] 16}} [CO-3,5-Ph (SO 3 Na) 2] 32 production dendrimer ^{(BHALys [3 H-Lys]} 16 [NH 2 .TFA] 32) (2.12g, 0.27mmol PyBOP (9.56 g, 18.37 mmol) was added to a stirred solution of DMF / DMSO (1: 1) (200 mL). A solution of 3,5-disulfobenzoic acid (5.39 g, 19.11 mmol) and diisopropylethylamine (12.2 mL, 70.02 mmol) in DMF / DMSO (1: 1) (150 mL) was added slowly. A sticky precipitate formed. The precipitate was removed and redissolved in DMSO and returned to the reaction. The mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into water (3.5 L) and filtered through a 0.45 micron filter.

Purification was performed by tangential flow filtration on Centramate (3K membrane, 2L sample reservoir). After the initial wash with Milli-Q water (18 L), the retentate is washed 3 times with 1 M sodium carbonate (100 mL) with Milli-Q water wash (1 L) in between, then the pH of the filtrate is medium Filtration continued until it became sex (about 20 L). The retentate was concentrated under reduced pressure and lyophilized to give the desired product as an off-white solid (2.34 g, 61%).
1H nmr (300 MHz, D ₂ O) λ (ppm): 1.0-2.0 (186H); 2.8-3.4 (62H); 4.0-4.4 (31H); 5.9 (1H); 7.0-7.3 (10H); 8.1-8.3 (96H).
LC / MS (ion-pair): ESI (-ve) m / z = 740.83 ((M-17H) 17-); 699.94 ((M-18H) 18-); 662.83 ((M-19H) 19-); 629.99 ((M-20H) ^20- ); 599.53 ((M-21H) ^21- ). MW = 12614 (M-, H-type) was obtained by deconvolving the data using the maximum entropy method. Calculated value (H type) (C ₄₂₃ H ₅₁₃ N ₆₃ O ₂₅₅ S ₆₄ ) 12612 (M-) Rf (min) = 3.14.
CE (pH 3): 98.6% Rf (min) = 10.97
CE (pH 9): 97.6% Rf (min) = 12.90

^{_{BHALys [3 H-Lys] 8}} [CO-4-Ph (SO 3 Na)] 16 manufacturing dendrimer ^{(BHALys [3 H-Lys]} 8 [NH 2 .TFA] 16) (68mg, 17.3μmol) in DMF ( 5 mL) To the stirred solution, PyBOP (303 mg, 0.58 mmol) was added. A solution of 4-sulfobenzoic acid monosodium salt (124 mg, 0.55 mmol) and diisopropylethylamine (386 μL, 2.22 mmol) in DMSO / DMF (5 mL / 10 mL) was slowly added. The mixture was stirred at room temperature under argon for 24 hours, then poured into water (200 mL) and filtered through a 0.45 micron filter. Purification was performed with Minimate (1K membrane, 250 mL sample reservoir) by tangential flow filtration and washed with water (1.5 L). The solvent was reduced from the retentate under reduced pressure. The product was redissolved in water (5 mL) and applied to a Sephadex size exclusion column (LH20, eluent: water) to collect fractions 1-8. The combined fractions were concentrated under reduced pressure, passed through an ion exchange column (69F, Na +) and lyophilized to give the desired product BHALys [ ³ H-Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ was obtained as a white solid (20 mg, 22%).
1H nmr (300 MHz, D ₂ O) λ (ppm): 1.0-1.9 (90H, CH2); 2.8-3.3 (30H, CH2); 4.0-4.4 (15H, CH); 5.9 (1H, CH); 7.0- 7.2 (10H, Ar-H); 7.5-7.8 (64H, Ar-H).
MS (direct injection): ESI (-ve) m / z = 5406 (MH)-(M-, sodium type)
Calculated value (sodium type) (C215H257N31O79Na16S16) 5404 (M-).
CE (pH 7): 91% Rf (min) = 10.51

^{_{BHALys [3 H-Lys] 16}} [CO-4-Ph (SO 3 Na)] 32 production dendrimer ^{(BHALys [3 H-Lys]} 16 [NH 2 .TFA] 32) (100mg, 13μmol) in DMSO (8 mL ) To the stirred solution, PyBOP (448 mg, 0.86 mmol) was added. A solution of 4-sulfobenzoic acid monopotassium salt (197 mg, 0.82 mmol) and diisopropylethylamine (571 μL, 3.28 mmol) in DMSO (12 mL) was added slowly. The mixture was stirred at room temperature for 24 hours under argon. The reaction mixture was poured into water (200 mL) and filtered through a 0.45 micron filter. Purification was performed by Stirred Cell (5K membrane, 250 mL sample reservoir) by tangential flow filtration, and this was washed with water (1.5 L). The solvent was reduced from the retentate under reduced pressure. The product is redissolved in water (5 mL), passed through an ion exchange column (69F, Na +) and lyophilized to yield the desired product BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na) ] ₃₂ was obtained as a white solid (89 mg, 65%).
1H nmr (300 MHz, D ₂ O) λ (ppm): 1.0-1.9 (186H, CH2); 2.8-3.2 (62H, CH2); 4.0-4.4 (31H, CH); 5.9 (1H, CH); 7.0- 7.2 (10H, Ar-H); 7.5-7.8 (128H, Ar-H).
MS (direct injection): ESI (-ve) m / z = 10756 (MH)-(M-, sodium type)
Calculated value (sodium type) (C423H481N63O159Na32S32) 10754 (M-).
CE (pH 9): 98% Rf (min) = 12.17

^{_{BHALys [3 H-Lys] 16}} [COCH 2 CH 2 (CO 2 Na)] 32 production dendrimer ^{(BHALys [3 H-Lys]} 16 [NH 2 .TFA] 32) (32mg, 4.1μmol) and triethylamine (36 [mu] L , 0.26 mmol) in DMF (5 mL) was added to succinic anhydride (26 mg, 0.26 mmol). The mixture was stirred at room temperature for 24 hours under argon. The reaction mixture was poured into water (50 mL) and filtered through a 0.45 micron filter. Purification was performed with Minimate (1K membrane, 70 mL sample reservoir) by tangential flow filtration. After initial washing with NaHCO ₃ (saturated) (100 mL), the retentate was washed with water (1.5 L). The solvent was reduced from the retentate under reduced pressure. The desired product BHALys [ ³ H-Lys] ₁₆ [COCH ₂ CH ₂ (CO ₂ Na)] is obtained by re-dissolving the product in water, passing through an ion exchange column (69F, Na +) and lyophilizing. ₃₂ was obtained as a white solid (17 mg, 52%).
1H nmr (300 MHz, D ₂ O) λ (ppm): 1.0-1.8 (186H, CH2); 2.3-2.7 (128H, CH2); 2.9-3.2 (62H, CH2); 4.0-4.2 (31H, CH); 6.0 (1H, CH); 7.1-7.3 (10H, Ar-H).
MS (direct injection): ESI (-ve) m / z = 7360 (MH)-(M-, H type)
Calculated value (H type) (C327H513N63O127) 7359 (M-).
CE (pH 9): 96% Rf (min) = 13.02

（実施例８）

(Example 8)

Study of plasma clearance and biodistribution of anionic dendrimers The method used in this study is the same as that used in Example 5. BHALys [Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ (closed circle), BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na) after IV administration of 5 mg / kg to rats )] ₃₂ (hollow circle) and BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ (triangle), the plasma concentration-time profile is illustrated in FIG.

BHALys [Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ (black), BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ (light gray), BHALys [Lys ] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ (dark gray) or BHALys [Lys] ₁₆ [CO-CH ₂ CH ₂ (CO ₂ Na)] ₃₂ (white) IV administered 30 The biodistribution of injected ³ H after time is illustrated in FIG. Panel A-% of injected ³ H present for each organ. Panel B-% of ³ H injection present per gram of tissue.
Example 9

Size Exclusion Chromatography on the Biological Sample Obtained in Example 8 The method used in this study is the same as in Example 6. BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ and BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] in plasma and urine on a Superdex 75 column _{The 32} size exclusion profiles are illustrated in FIG.
(Example 10)

Tritium labeled PEG dendrimer
BHALys production of ^{_{[3 H-Lys] 8 [}} PEG 200] 16
To a stirred solution of BHALys [ ³ H-Lys] ₈ [NH ₂ .TFA] ₁₆ (125 mg, 0.03 mmol) in DMF (8 mL) was added PyBOP (556 mg, 1.0 mmol), and then PEG200 (240 mg, 1.0 mmol). mmol), N, N-diisopropylethylamine (709 μL, 4.0 mmol) in DMF (16 mL) and DMSO (2 mL) were added. The solution was stirred at room temperature for 16 hours. The reaction mixture was poured into water (180 mL), filtered and washed with water. The aqueous solution was transferred to a 3K stirring cell, water was passed through the cell, and the remaining water was removed by lyophilization, whereby BHALys [ ³ H-Lys] ₈ [PEG ₂₀₀ ] ₁₆ was free-flowing white solid (20 mg, 11 %).
LC / MS (Philic TFA): Rf (min) = 16.72. ESI (+ ve) m / z = 5598 (M + H ⁺ ).

BHALys production of ^{_{[3 H-Lys] 16 [}} PEG 200] 32
To a stirred solution of BHALys [ ³ H-Lys] ₁₆ [NH ₂ .TFA] ₃₂ (30 mg, 0.004 mmol) in DMF (3 mL) was added PyBOP (142 mg, 0.271 mmol) followed by PEG200 (62 mg 0.263 mmol), N, N-diisopropylethylamine (182 μL, 1.04 mmol) in DMF (3 mL) was added. The solution was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure and the resulting crude mixture was dissolved in a minimum amount of water. Purification on a Sephadex column (LH-20) with water as the eluent, after removal of the water by lyophilization, the desired product BHALys [ ³ H-Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ is obtained as a white solid (20 mg, 44%). LC / MS (Philic TFA): Rf (min) = 16.74. ESI (+ ve) m / z = 11,141 (M + H ⁺ ).

BHALys production of ^{_{[3 H-Lys] 16 [}} PEG 570] 32
BHALys [ ³ H-Lys] ₁₆ [NH ₂ .TFA] ₃₂ (20 mg, 0.003 mmol) in dry DMF (2 mL) was stirred under nitrogen with triethylamine (36 μL, 0.261 mmol) and PEG685.75, NHS. The ester (119 mg, 0.174 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was poured into a 5K stirred cell, water (600 mL) was passed through the cell, and the remaining water was removed by lyophilization (twice) to remove BHALys [ ³ H-Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ in glass. Obtained as a solid (50 mg, 88%). LC (Philic TFA): Rf (min) 12.42.

BHALys production of ^{_{[3 H-Lys] 8 [}} PEG 2KD] 16
PYBOP (141 mg, 0.271 mmol) was added to a stirred solution of BHALys [ ³ H-Lys] ₈ [NH ₂ .TFA] ₁₆ (30 mg, 0.008 mmol) in dry DMF (2 mL) under nitrogen, then PEG2000 , NHS ester (612 mg, 0.306 mmol), N, N-diisopropylethylamine (180 μL, 1.04 mmol) in DMF (1.4 mL) and DMSO (0.6 mL) were added. The solution was stirred at room temperature for 16 hours. The reaction mixture was poured into a 10K stirred cell, water (800 mL) was passed through the cell, and the remaining water was removed by lyophilization, whereby BHALys [ ³ H-Lys] ₈ [PEG _2KD ] ₁₆ was free-flowing white solid. (149 mg, 54%).

BHALys production of ^{_{[3 H-Lys] 16 [}} PEG 2KD] 32
To a stirred solution of BHALys [ ³ H-Lys] ₁₆ [NH ₂ .TFA] ₃₂ (30 mg, 0.004 mmol) in dry DMF (2 mL) was added PyBOP (142 mg, 0.272 mmol), and then PEG2000 , NHS ester (522 mg, 0.261 mmol), N, N-diisopropylethylamine (182 μL, 1.04 mmol) in DMF (3 mL) and DMSO (1 mL) were added. The solution was stirred at room temperature for 16 hours. The reaction mixture was poured into water, filtered and washed with water. Purification was performed with Mini-mate (10K membrane, 2 L of water) by tangential flow filtration. BHALys [ ³ H-Lys] ₁₆ [PEG _2KD ] ₃₂ was obtained as a free-flowing white solid (210 mg, 76%) by removing the solvent by lyophilization.
LC / MS (Philic TFA): Rf (min) = 16.29. ESI (+ ve) m / z = 67,696 (M + H ⁺ )

（実施例１１）

Example 11

Study of Plasma Clearance and Biodistribution of PEG Dendrimer The method used in this study is the same as that used in Example 5.

BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ (closed circle), BHALys [Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ (hollow circle), BHALys [Lys] ₈ [PEG ₂₀₀₀ ] ₁₆ (closed triangle) and BHALys [ The plasma concentration-time profile for Lys] ₁₆ [PEG ₂₀₀₀ ] ₃₂ (hollow triangle) is illustrated in FIG. Data on BHALys [Lys] ₈ [PEG ₂₀₀ ] ₁₆ are not shown. This is because the disappearance is very rapid and is hidden in the data related to BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ .

BHALys [Lys] ₁₆ [PEG ₂₀₀₀ ] ₃₂ (black bar, 7 days), BHALys [Lys] ₈ [PEG ₂₀₀₀ ] ₁₆ (gray bar, 5 days) and BHALys [Lys] ₁₆ [PEG ₅₇₀ ] after IV administration _The biodistribution of ₃₂ (white bar, 30 hours) is illustrated in FIG.
Example 12

Size Exclusion Chromatography on the Biological Sample Obtained in Example 11 The method used in this study is the same as in Example 6. ³ H-labeled BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ (Panel A; t0, hollow square, t = 1 hour, closed circle, t = 4 hour, hollow in plasma after IV administration of 5 mg / kg (Circle), BHALys [Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ (panel B; 24 hours), BHALys [Lys] ₈ [PEG ₂₀₀₀ ] ₁₆ (panel C; 48 hours) and BHALys [Lys] ₁₆ [PEG ₂₀₀₀ ] ₃₂ ( For panel D; 48 hours), the size exclusion profile on a Superdex 75 column is illustrated in FIGS.

BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ (Panel A; 0 to 4 hours urine, closed circle, 8 to 24 hours urine, hollow circle) and BHALys [Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ (Panel B; 8 16A and B illustrate the size exclusion profile of ³ H excreted in urine after IV administration (˜24 hours urine). Arrows indicate complete dendrimer retention time.

The abstract applicant has studied ³ H-labeled poly-L-lysine dendrimers (here, both BHALys [Lys] ₈ [NH ₂ ] ₁₆ or BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ are produced on the surface Was left without capping). For comparison, a third dendrimer, namely BHALys [Lys] ₈ [NH ₂ ] ₁₆ core, is completely capped with D-lysine and Lys ₁₆ dendrimer BHALys [Lys] ₈ with D-lysine in its outer layer. What formed [D-Lys] ₁₆ [NH ₂ ] ₃₂ was also examined. In both cases, the dendrimer was covered with cationic amine groups at physiological pH. Plasma clearance and biodistribution were determined for these substances (Example 5). The in vivo fate of ³ H dendrimers was examined in more detail by separating different radiolabeled species present in plasma by size exclusion chromatography (Example 6). The data shows that poly-L-lysine dendrimers are rapidly removed from plasma after intravenous administration, but are subsequently metabolized and re-incorporated into the endogenous resynthesis process.

After intravenous administration, both BHALys [Lys] ₈ [NH ₂ ] ₁₆ and BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ are removed from plasma very rapidly, showing an initial plasma half-life of less than 10 minutes (Figure 2). This rapid initial loss is not dose-dependent (Figure 3), and when examining the whole blood profile (not plasma) (Figure 4), the L-lysine surface group is changed to D-lysine. (Figure 5) was also obvious. These relatively high molecular weight species have a surprisingly high initial distribution volume (V _c ) with higher molecular weights of the 4th generation dendrimers BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ and BHALys [Lys] ₈ [D- Lys] ₁₆ [NH ₂ ] ₃₂ had a higher V _c than the three generations with lower molecular weights. Thus, the V _c values for the 3rd and 4th generation dendrimers appear to be more highly correlated with surface charge than molecular weight. This data is consistent with an initial “distribution” process that reflects the rapid binding of polycationic dendrimers to the vascular endothelium, which occurs as a process driven by electrostatic interactions and resulting in loss from circulating plasma. Yes. In contrast, it does not appear to be a typical extravasation process. This is because it is difficult for such highly charged polymers to cross the vascular endothelium quickly, and it is expected to increase with decreasing molecular weight and surface charge, but in fact the opposite phenomenon was observed. It is. These trends were also evident in whole blood pharmacokinetics. However, for small dendrimers, the C _p ⁰ value was about 1/2 (and the corresponding V _c value was doubled). This suggests that the 3rd generation dendrimer with relatively low charge had less dendrimer binding to erythrocytes.

  In summary, uncapped poly-L-lysine dendrimers are rapidly removed from plasma upon intravenous injection, and radiolabels that were originally associated with complete dendrimers are now free lysine and some The data showed that it reappears in plasma associated with molecular species co-eluting with larger molecular weight (possibly proteinaceous) substances (including albumin). This data also shows that highly charged cationic dendrimers quickly bind to the endothelial cell surface immediately after injection and are then hydrolyzed to yield circulating free lysine, which ultimately leads to the protein biosynthetic pathway It is also suggested that it will be taken in again. These data are, as far as we know, the first data describing the biodegradation and resorption of poly-L-lysine dendrimers in vivo. Therefore, by appropriately manipulating the surface properties of poly-L-lysine dendrimers, the initial residence time in plasma can be increased. Thus, uncapped poly-L-lysine surfaces can be drug delivery systems based on biodegradable and bioabsorbable dendrimers.

  In a separate study, capping the surface of poly-L-lysine dendrimer cores with anionic aryl sulfonate or alkylcarboxylate groups affects the pharmacokinetics and biodistribution pattern of dendrimers after intravenous administration to rats The effect was also investigated.

Two different size dendrimer cores BHALys [Lys capped with benzene sulfonate (CO-4-Ph (SO ₃ Na)) or benzene disulfonate (CO-3,5-Ph (SO ₃ Na) ₂ ) end groups ] ₈ [NH ₂ ] ₁₆ and BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ (with 8 and 16 lysine groups on the outer layer, respectively) make it easy to influence the size and surface charge of dendrimers It was made possible to distinguish (Synthesis Example 7). Four tritium-labeled lysine dendrimers BHALys [Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ ; BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ ; BHALys [Lys] ₁₆ [ CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ and BHALys [Lys] ₁₆ [CO-CH ₂ CH ₂ (CO ₂ Na)] ₃₂ were administered intravenously (5 mg / kg), plasma, urine And the radioactivity in the stool was monitored over 30 hours (Example 8). Animals were sacrificed 30 hours after administration, major organs were removed, homogenized, and radiolabeled assayed.

The elimination half-life of all four dendrimers was essentially the same (about 1 hour), but the plasma clearance and volume of distribution of BHALys [Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ was Lys ₁₆ A higher than dendrimer was shown by plasma concentration-time profile. About 30% of the injected radiolabel associated with BHALys [Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ and BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ dendrimers Is excreted in the urine 30 hours after administration, whereas the highly charged BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ Only 3% of the dose during the period was eliminated in the urine.

The clearance profile of succinate dendrimers was quite different from other anionic dendrimers, and succinate dendrimers were cleared from plasma very rapidly upon intravenous infusion. The initial volume of distribution (V _c ) was also higher than other anionic dendrimers and close to that of cationic dendrimers, suggesting an initial interaction with blood components or endothelial surfaces that rapidly remove radiolabel from plasma (Figure Four). The initial loss rate in the first 20 minutes in plasma was also very rapid and again occurred on a time scale similar to that seen with cationic dendrimers. After the initial rapid decline, the radiolabel removal rate appeared to be reduced, leaving a quantifiable amount of radiolabel 30 hours after administration. It is not known whether this second slow removal rate reflects the redistribution of label to plasma as previously seen in the cationic system. Anionic dendrimers appeared to be almost completely eliminated from erythrocytes. In contrast to other anionic dendrimers, most of the injected radiolabel associated with succinate dendrimers is excreted in the urine (63.71 ± 8.98%), while pooled feces are very small A quantifiable amount of radiolabel was recovered (1.21 ± 0.97%) (Table 3).

Of plasma samples from rats administered BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ and BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ Size exclusion chromatography revealed that both anionic dendrimers bind rapidly to plasma components to form high molecular weight species (<67 kDa). Although no degradation products were identified in plasma, radiolabels in urine were primarily associated with molecular species close to the molecular weight of Lys-aryl sulfonate monomers. Interestingly, most of the radiolabel in the urine was excreted on a time scale (8-24 hours after administration) with very low levels of plasma radioactivity. The organ deposition pattern revealed that the residual radioactivity present 30 hours after administration of each dendrimer was concentrated mainly in the liver, spleen and kidney. In the case of BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ , the recovery of radioactivity was particularly high in the liver (about 50% of the dose). This data suggests that metabolism occurs after the benzenesulfonate dendrimer is cleared from the plasma, which subsequently facilitates urinary elimination of degradation products. In contrast, the low recovery of radioactivity from BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ in urine is probably a result of increased surface charge density of dendrimers As such, it reflects the reduced sensitivity to metabolism, which may result in high recovery in the liver.

  Thus, the plasma clearance of anionic dendrimers is slower than that seen previously for uncapped cationic poly-L-lysine dendrimers. Second, within the anion series, plasma clearance is primarily determined by dendrimer size rather than surface charge. Ultimately, however, the surface charge determines the pattern of renal exclusion and biodistribution and may be attributed to the difference in sensitivity to metabolism of benzene sulfonate capped dendrimers and benzene disulfonate capped dendrimers.

PEGylation is known to reduce protein recognition by proteolytic enzymes and suppress phagocytic removal of proteins and colloids, resulting in increased plasma circulation time. Therefore, in this application, the effect of PEGylation (Synthesis Example 10) on the plasma profile, biodistribution pattern and urinary exclusion of ³ H-labeled poly-L-lysine dendrimer was investigated after intravenous administration of 5 mg / kg dendrimer. Investigated in rats (Pharmacokinetics and Biodistribution Example 11). In general, the plasma half-life of PEGylated dendrimers and the extent of urinary exclusion depend on molecular weight, whereas large PEGylated dendrimers (ie> 30 kDa) were removed from plasma relatively slowly (t _1/2 1 to 3 days), small molecular species (ie <20 kDa) were rapidly cleared from plasma into urine (t _1/2 1-10 hours). Large dendrimers seemed to eventually concentrate in the reticulo-organs (liver and spleen), but this occurred over time and the degree of absolute accumulation was low (<10% of the dose) ). In the SEC, dendrimers derivatized with the smallest (200Da) PEG chain showed some signs of interaction with plasma components that resulted in the generation of apparently higher molecular weight species, but the urinary exclusion was It was very rapid and only complete dendrimers were recovered in the urine. Dendrimers derivatized with larger PEGylated species (ie 2000 Da) existed as parent (unchanged) dendrimers in plasma and urine. Therefore, the size of PEGylated poly-L-lysine dendrimer complexes can be manipulated to optimally adjust their pharmacokinetics.

Example of differentially protected intermediate and partial PEG structure (Example 15)

Production of _{BHALys [Lys] 8 [α-} Boc] 8 [ε-NH 2] 8
i. Production of _{BHALys [Lys] 8 [α-} Boc] 8 [ε-CBz] 8
A solution of BHALys [Lys] ₄ [NH ₂ .TFA] ₈ (1.59 mmol), triethylamine (4.50 ml, 32.30 mmol) and DMF (30 ml) was added to a magnetically stirred solution of solid PNPO-α-Boc-ε-CBz. -Lys (7.75 g, 15.45 mmol) was added in one portion at room temperature. The reaction suspension immediately turned light yellow and the active ester was completely dissolved after stirring for about 5 minutes. Stirring was continued at room temperature for a further 22 hours. The crude reaction mixture was poured into a large beaker containing ice water and a fine yellow precipitate formed. The suspension was filtered and the resulting retained solid was air dried overnight with suction. The resulting dry pale yellow cake was pulverized to a fine powder and resuspended in acetonitrile. The suspension was stirred at room temperature for 30 minutes and then filtered. The retained solid was air dried once more, ground again, resuspended in acetonitrile, filtered, and air dried overnight to give BHALys [Lys] ₈ [α-Boc] ₈ [ε-CBz] ₈ t ( 5.52 g, 87%) was obtained as a colorless solid.
LC / MS (Phobic / TFA): ESI (+ ve) found [M + H / 3] + m / z = 1328; calculated (as C207H305N31O47) 3979.9; Rf (min) = 20.22 min.

ii. Production of _{BHALys [Lys] 8 [α-} Boc] 8 [ε-NH 2] 8
BHALys [Lys] ₈ [α-Boc] ₈ [ε-CBz] ₈ (500 mg, 0.126 mmol) is suspended in 9: 1 DMF / H ₂ O (12.5 ml) and ammonium formate (127 mg, 2.01 mmol) is added. After stirring for 5 minutes, Pd / C (10% w / w, 266 mg) was added and stirring was continued for 2 hours. The reaction was stopped by filtering off the catalyst and the filter was washed with 9: 1 DMF / H ₂ O (10 ml) followed by water (2 ml). The combined filtrate was concentrated under reduced pressure to give a colorless syrup. It is treated with water (10 ml), the water is removed under reduced pressure, and then lyophilized from water to give BHALys [Lys] ₈ [α-Boc] ₈ [ε-NH ₂ ] ₈ to a fine white Obtained as a lyophilizate (155 mg, 42%).
LC / MS (Hydrophilic / TFA): ESI (+ ve) m / z = 969.83 [M + 3H] / 3 +, 727.67 [M + 4H] / 4 +, 582.39 [M + 5H] / 5 +; calculation Value (C143H257N31O31) 2906.8 g / mol. By deconvolving the data using a conversion operation, mw = 2906.5 was obtained. Rf (minutes) = 14.7.
(Example 16)

Production of _{BHALys [Lys] 8 [α-} NH 2 -TFA] 8 [ε-CBz] 8
BHALys [Lys] ₈ [α-Boc] ₈ [ε-CBz] ₈ (1000 mg, 0.251 mmol) was suspended in acetic acid (5.5 ml), and trifluoroacetic acid (5.5 ml) was added dropwise with stirring at 0 ° C. . The reaction mixture was warmed to room temperature and left to stir for 17 hours, at which time the reaction mixture was triturated in diethyl ether. The resulting suspension was stirred for 10 minutes and the liquid was removed by centrifugation and decanting. The remaining precipitate is washed by stirring with diethyl ether for 10 minutes and the diethyl ether is again removed by centrifugation and decantation, then the precipitate is dried under reduced pressure, dissolved in water and lyophilized. Gave BHALys [Lys] ₈ [α-NH ₂ .TFA] ₈ [ε-CBz] ₈ as a white powder (840 mg, 105%).
LC / MS (Hydrophilic / TFA): ESI (+ ve) m / z = 1060.70 [M + 3H] / 3 +, 795.51 [M + 4H] / 4 +, 636.30 [M + 5H] / 5 +; calculation Value (C167H241N31O31) 3178.95 g / mol. By deconvolving the data using a conversion operation, mw = 3178.0 was obtained. Rf (minutes) = 19.1.
(Example 17)

BHALys [Lys] ₁₆ production of _{[α-Boc] 16 [ε} -NH 2] 16
i. _{BHALys [Lys] 16 [α-} Boc] 16 [ε-CBz] 16 production of
To a stirred solution of BHALys [Lys] ₈ [NH ₂ .TFA] ₁₆ (0.81 mmol), triethylamine (4.50 ml, 32.30 mmol) and DMF (30 ml), solid PNPO-α-Boc-ε-CBz- Lys (7.94 g, 15.83 mmol) was added in one portion at room temperature. The reaction suspension immediately turned light yellow and the active ester was completely dissolved after stirring for about 5 minutes. Stirring was continued at room temperature for a further 22 hours. The crude reaction mixture was poured into a large beaker containing ice water and a fine yellow precipitate formed. The suspension was filtered and the resulting retained solid was air dried overnight with suction. The resulting dry pale yellow cake was pulverized to a fine powder and resuspended in acetonitrile. The suspension was stirred at room temperature for 30 minutes and then filtered. The retained solid was air-dried overnight to yield BHALys [Lys] ₁₆ [α-Boc] ₁₆ [ε-CBz] ₁₆ (5.79 g, 91%) as a colorless solid.
LC / MS (Phobic / TFA): ESI (+ ve) measured value [M + H / 4] + m / z = 1977; [M + H / 5] + m / z = 1582; calculated value 7904.9 (as C407H609N63O95); Rf (min) = 23.51 min.

ii. BHALys [Lys] ₁₆ production of _{[α-Boc] 16 [ε} -NH 2] 16
A suspension of BHALys [Lys] ₁₆ [α-Boc] ₁₆ [ε-CBz] ₁₆ (50 mg, 0.006 mmol), 10% Pd / C (53 mg) and acetic acid (2 ml) at room temperature under hydrogen for 16 hours Stir vigorously. The black suspension was filtered. The filtrate was concentrated under reduced pressure to give a straw oil product (26 mg, 71%).
LC / MS (Phobic / TFA): ESI (+ ve) measured [M + H / 5] + m / z = 1152; [M + H / 6] + m / z = 961; [M + H / 7] + m / z = 824; [M + H / 8] + m / z = 721; [M + H / 9] + m / z = 641; calculated value (as C279H513N63O63) 5758.53; Rf ( Minutes) = 2.37 minutes.
(Example 18)

_{BHALys [Lys] 16 [α-} NH 2 .TFA] 16 [ε-CBz] 16 production of
BHALys [Lys] ₁₆ [α-Boc] ₁₆ [ε-CBz] ₁₆ (1000 mg, 0.127 mmol) was suspended in acetic acid (5.5 ml), and trifluoroacetic acid (5.5 ml) was added dropwise with stirring at 0 ° C. . The reaction mixture was warmed to ambient temperature and left stirring for 17 hours. The reaction mixture was triturated in diethyl ether (150 ml) and the resulting suspension was stirred for 10 minutes. The liquid was removed by centrifugation (4000 rpm, 10 minutes) and decantation, the remaining precipitate was washed by stirring with diethyl ether (150 ml) for 10 minutes, and the diethyl ether was again removed by centrifugation and decantation. The precipitate was dried under reduced pressure, dissolved in water (50 mL) and lyophilized to give BHALys [Lys] ₁₆ [α-NH ₂ .TFA] ₁₆ [ε-CBz] ₁₆ as a white powder (832 mg, 114 %).
LC / MS (Hydrophilic / TFA): ESI (+ ve) m / z = 1576.85 [M + 4H] / 4 +, 1261.34 [M + 5H] / 5 +, 1051.27 [M + 6H] / 6 +, 901.15 [M + 7H] / 7 +; calculated (as C327H481N63O63) 6302.83 g / mol. By deconvolving the data using a conversion operation, mw = 6301.5 was obtained. Rf (minutes) = 19.0.
Example 19

Production of _{BHALys [Lys] 8 [α-} NH 2] 8 [ε-Boc] 8
i. Production of _{BHALys [Lys] 8 [α-} CBz] 8 [ε-Boc] 8
A solution of BHALys [Lys] ₄ [NH ₂ .TFA] ₈ (1.59 mmol), triethylamine (4.40 ml, 31.57 mmol) and DMF (32 ml) was added to a magnetically stirred solution of solid PNPO-α-CBz-ε-Boc. -Lys (7.69 g, 15.33 mmol) was added in one portion at room temperature. The reaction suspension immediately turned light yellow and the active ester was completely dissolved after stirring for about 5 minutes. Stirring was continued for an additional 19 hours at room temperature. The crude reaction mixture was poured into a large beaker containing acetonitrile (about 300 ml). The suspension was filtered and the resulting retained solid was air dried overnight with suction. The resulting dry pale yellow cake was ground to a fine powder (mortar and pestle) and resuspended in acetonitrile (400 ml). The suspension was magnetically stirred at room temperature for 60 minutes and then filtered. The retained solid was air-dried again, ground again, resuspended in acetonitrile, filtered, and air-dried overnight to obtain BHALys [Lys] ₈ [α-CBz] ₈ [ε-Boc] ₈ (5.41 g, 85%) was obtained as an off-white solid.
LC / MS (Phobic / TFA / Speedy Ramp): ESI (+ ve) measured value [M + H / 3] + m / z = 1328; calculated value (as C207H305N31O47) 3979.9; Rf (min) = 12.98 minutes.

ii. Production of _{BHALys [Lys] 8 [α-} NH 2] 8 [ε-Boc] 8
BHALys [Lys] ₈ [α-CBz] ₈ [ε-Boc] ₈ (5.0 mg, 1.26 μmol) was suspended in 9: 1 DMF / H ₂ O (2 ml), stirred at ambient temperature, and ammonium formate (2.5 mg, 40.2 μmol) was added followed by Pd / C (10% w / w, 2.7 mg) and stirring was continued for 20 hours. The reaction was stopped by filtering off the catalyst and the filter was rinsed with 9: 1 DMF / H ₂ O (1 ml). The filtrate was concentrated under reduced pressure to give a colorless syrup. It is treated with water (1 ml), the water is removed under reduced pressure, and lyophilized from water (1 ml) to give BHALys [Lys] ₈ [α-NH ₂ ] ₈ [ε-Boc] ₈ Obtained as a fine white lyophilizate (2 mg, 42%).
LC / MS (hydrophilic / TFA): ESI (+ ve) m / z = 969.88 [M + 3H] / 3 +, 727.56 [M + 4H] / 4 +; calculated value (as C143H257N31O31) 2906.8 g / mol. By deconvolving the data using a conversion operation, mw = 2906.5 was obtained. Rf (minutes) = 17.2.
(Example 20)

Production of _{BHALys [Lys] 8 [α-} CBz] 8 [ε-NH 2 .TFA] 8
BHALys [Lys] ₈ [α-CBz] ₈ [ε-Boc] ₈ (20.0 mg, 0.005 mmol) was suspended in acetic acid (109 μl) and stirred in a water bath. Trifluoroacetic acid (109 μl) was carefully added to dissolve all solids and stirring was continued for 16 hours at ambient temperature. The reaction was stopped by removing all volatiles under reduced pressure to give a clear and colorless oil. This oil was triturated in diethyl ether and the resulting white precipitate was washed with diethyl ether and dried under reduced pressure to give BHALys [Lys] ₈ [α-CBz] ₈ [ε-NH ₂ . TFA] ₈ (12.1 mg, 76%) was obtained as a white solid.
LC / MS (Hydrophilic / TFA): ESI (+ ve) m / z = 1060.43 [M + 3H] / 3 +, 795.63 [M + 4H] / 4 +, 636.79 [M + 5H] / 5 +; calculation Value (as C167H241N31O31) 3178.95 g / mol. By deconvolving the data using a conversion operation, mw = 3179.25 was obtained. Rf (minutes) = 16.6.
(Example 21)

_{BHALys [Lys] 16 [α-} NH 2] 16 [ε-Boc] 16 production of
i. BHALys [Lys] ₁₆ production of _{[α-Fmoc] 16 [ε} -Boc] 16
To a stirred solution of BHALys [Lys] ₈ [NH ₂ .TFA] ₁₆ (0.54 mmol), DIPEA (3.0 ml, 17.22 mmol) and DMF (11 ml), solid pentafluorophenyloxy-α-Fmoc-ε -Boc-Lys (6.58 g, 10.36 mmol) was added in one portion at room temperature. The reaction suspension immediately turned light yellow and after about 10 minutes of stirring, the active ester was completely dissolved. Stirring was continued at room temperature for a further 18 hours. After that, the crude reaction mixture became so thick that magnetic stirring could no longer be continued. Acetonitrile (about 300 ml) was added to the reaction flask and the solid mass was crushed with a spatula until it was sufficient to resume stirring (1 hour). The suspension was filtered and air dried overnight under reduced pressure. The resulting almost colorless cake was ground with a mortar and pestle and the fine solid was resuspended in acetonitrile (500 ml) for 2 hours. The suspension was then filtered and a colorless solid was collected and air dried at room temperature overnight. The expected product was obtained as an off-white solid (4.72 g, 94%). This product was characterized as the Fmoc deprotected derivative BHALys [Lys] ₁₆ [α-NH ₂ ] ₁₆ [ε-Boc] ₁₆ (see ii).

ii. _{BHALys [Lys] 16 [α-} NH 2] 16 [ε-Boc] 16 production of
To a magnetically stirred suspension of BHALys [Lys] ₁₆ [α-Fmoc] ₁₆ [ε-Boc] ₁₆ (1.0 g, 0.108 mmol) and DMF (10 ml), add neat piperidine (1 ml, 10.11 mmol). Added in one portion at room temperature. The suspension was stirred for an additional 17 hours and the crude reaction mixture became a pale yellow solution. The mixture was concentrated under reduced pressure to give an off-white solid that was then suspended in diethyl ether (ca. 100 ml). After stirring at room temperature for 30 minutes, the suspension was filtered and the collected solid was air dried overnight. The expected product was obtained as a colorless solid (0.60 g, 97%).
LC / MS (Phobic / TFA): ESI (+ ve) found [M + H / 4] + m / z = 1441; [M + H / 5] + m / z = 1153; [M + H / 6] + m / z = 961; calculated (as C279H513N63O63) 5758.5; Rf (min) = 2.95 min.
(Example 22)

_{BHALys [Lys] 16 [α-} Boc] 16 production of [ε-NH ₂ Boc] _{16
BHALys [Lys] 16 [α-} Boc] 16 [ε-Fmoc] 16 production and _{BHALys [Lys] 16 [α-} Boc] 16 [ε-Fmoc] 16 production of
To a stirred mixture of BHALys [Lys] ₁₆ [α-CBz] ₁₆ [ε-Boc] ₁₆ (0.01 mmol) and dichloromethane (0.5 ml), neat TFA (0.5 ml) was added dropwise under nitrogen. Stirring was continued at room temperature for 18 hours. Volatile reaction components were removed under reduced pressure and the resulting rubbery residue was treated with diethyl ether to induce precipitation of the salt product. The suspension was filtered and the collected solid was washed with diethyl ether. The resulting solid was dissolved in water and concentrated to dryness by lyophilization overnight. The product was obtained as a fluffy colorless solid.
LC / MS (hydrophilic / TFA): ESI (+ ve) found [M + H / 4] + m / z = 1579; [M + H / 5] + m / z = 1262. Calculated (as C ₃₂₇ H ₄₈₁ N ₆₃ O ₆₃ ) 6302.8.
(Example 23)

_{BHALys [Lys] 16 [α-} CBz] 16 [ε-NH 2 .TFA] 16 production of
To a stirred mixture of BHALys [Lys] ₁₆ [α-CBz] ₁₆ [ε-Boc] ₁₆ (0.01 mmol) and dichloromethane (0.5 ml), neat TFA (0.5 ml) was added dropwise under nitrogen. Stirring was continued at room temperature for 18 hours. Volatile reaction components were removed under reduced pressure and the resulting rubbery residue was treated with diethyl ether to induce precipitation of the salt product. The suspension was filtered and the collected solid was washed with diethyl ether. The resulting solid was dissolved in water and concentrated to dryness by lyophilization overnight. The product was obtained as a fluffy colorless solid.
LC / MS (hydrophilic / TFA): ESI (+ ve) found [M + H / 4] + m / z = 1579; [M + H / 5] + m / z = 1262. Calculated (as C ₃₂₇ H ₄₈₁ N ₆₃ O ₆₃ ) 6302.8.
(Example 24)

_{BHALys [Lys] 16 [α,} α-Boc] 8 [α, ε-Boc] 8 [ε, α-Boc] 8 [ε, ε-CBz] Production of ₈
i. _{BHALys [Lys] 8 [ε-} CBz] 8 [α-Lys] 8 [Boc] 16 production of
To a stirred solution of BHALys [Lys] ₈ [ε-CBz] ₈ [α-NH ₂ .TFA] ₈ (3 g, 0.75 mmol) in DMF (30 mL), DBL-OPNP (3.6 g, 7.2 mmol) and triethylamine (2.1 mL, 15 mmol) was added. The resulting yellow solution was stirred at room temperature for 16 hours. The reaction mixture was added to stirred acetonitrile (300 mL), resulting in a white precipitate in the yellow solution. The precipitate was collected by filtration and washed with acetonitrile to remove residual colored material. Next, the precipitate was dried at room temperature under reduced pressure to obtain BHALys [Lys] ₈ [ε-CBz] ₈ [α-Lys] ₈ [Boc] ₁₆ as a white powder (4.2 g, 98%).
LC / MS (Fast Hydrophobic / TFA): Rf (min) = 13.70; ESI (+ ve) m / z = 1936 ([M + 3] / 3), 1452 ([M + 4] / 4), 1062 ([M + 5-Boc] / 5); calculated value C295H465N47O71. M + 1. 5083.4.

ii. _{BHALys [Lys] 8 [ε-} NH 2] s [α -Lys] 8 [Boc] 16 production of
To a stirred solution of BHALys [Lys] ₈ [ε-CBz] ₈ [α-Lys] ₈ [Boc] ₁₆ (2.2 g, 0.38 mmol) in acetic acid (30 mL), 10% Pd / C (101 mg, 0.095 mmol ) Was added. The resulting homogeneous mixture was placed under hydrogen for 16 hours at room temperature. The solution was filtered and concentrated under reduced pressure. The viscous residue obtained was redissolved in water and lyophilized to obtain BHALys [Lys] ₈ [ε-NH ₂ ] ₈ [α-Lys] ₈ [Boc] ₁₆ (1.97 g, 0.38 mmol). Obtained. This material contained some acetic acid residue.
LC / MS (hydrophilic / TFA): Rf (min) = 18.53; ESI (+ ve) m / z = 1184 ([M + 4] / 4), 947 ([M + 5] / 5), 790 ( [M + 6] / 6); calculated. C231H417N47O55. M + 1. 4731.1.

iii. _{BHALys [Lys] 16 [α,} α-Boc] 8 [α, ε-Boc] 8 [ε, α-Boc] 8 [ε, ε-CBz] Production of ₈
To a stirred solution of BHALys [Lys] ₈ [ε-NH ₂ ] ₈ [α-Lys] ₈ [Boc] ₁₆ (90 mg, 0.019 mmol) in DMF (10 mL), PNPO-α-Boc-ε-CBz- Lys (90 mg, 0.18 mmol) and triethylamine (0.05 mL, 0.35 mmol) were added. The resulting yellow solution was stirred at room temperature for 16 hours. The reaction mixture was then added to stirred acetonitrile (100 mL), resulting in a white precipitate in the yellow solution. The precipitate was collected by filtration and washed with acetonitrile to remove residual colored material. By drying the precipitate at room temperature under reduced pressure, BHALys [Lys] ₁₆ [α, α-Boc] ₈ [α, ε-Boc] ₈ [ε, α-Boc] ₈ [ε, ε-CBz] ₈ (51 mg, 35%) was obtained.
LC / MS (hydrophobic TFA Speedy Rp): Rf (min) = 14.32; ESI (+ ve) m / z = 2544 ([M + 3] / 3), 1909 ([M + 4] / 4), 1527 ([M + 5] / 5); calculated. C383H625N63O95. M + 1.
(Example 25)

_{BHALys [Lys] 16 [α,} α-Boc] 8 [α, ε-Boc] 8 [ε, α-Boc] 8 [ε, ε-Fmoc] Production of ₈
To a stirred solution of BHALys [Lys] ₈ [ε-NH ₂ ] s [α-Lys] ₈ [Boc] ₁₆ (100 mg, 0.017 mmol) in DMF (10 mL), PFP-Lys-α-Boc-ε- Fmoc (96 mg, 0.15 mmol) and triethylamine (0.04 mL, 0.27 mmol) were added. The solution was stirred at room temperature for 16 hours. The reaction mixture was then added to acetonitrile (100 ml) resulting in a clear gelatinous precipitate. The precipitate was collected by filtration and washed with acetonitrile. By drying the precipitate at room temperature, BHALys [Lys] ₁₆ [α, α-Boc] ₈ [α, ε-Boc] ₈ [ε, α-Boc] ₈ [ε, ε-Fmoc] ₈ (30 mg, 21%).
LC / MS (hydrophobic / TFA): Rf (min) = 7.72; ESI (+ ve) m / z = 1667 ([M + 5] / 5), 1389 ([M + 6] / 6), 1191 ( [M + 7] / 7); calculated. C439H657N63O95. M + 1 8332.

Production of clearly defined PEG1.7KD
To a stirred solution of HO ₂ C-PEG ₁₁₄₆ -NH ₂ (245 mg, 0.21 mmol, 1.07 eq) and PEG ₅₇₀ -NHS (136 mg, 0.2 mmol) in DMF (6 mL) was added 2 mL of buffer (pH 8.5). , To a stirred solution of Na ₂ HPO ₄ (100 mL) was added 2.5 mL of 0.1 M HCl solution, and prepared by stirring for 5 minutes after the addition was complete. The reaction mixture was stirred at room temperature overnight. The solvent was removed by rotavap to give PEG ₁₇₁₆ -CO ₂ H as a residue. It was purified by LC to give the expected product (yield 80%).
(Example 26)

Production of _{BHALys [Lys] 8 (α-} NH 2 .TFA) 8 (ε-PEG 571) 8
i. Production of _{BHALys [Lys] 8 (α-} Boc) 8 (ε-PEG 571) 8
To a solution of BHALys [Lys] ₈ (α-Boc) ₈ (ε-NH ₂ .TFA) ₈ (9.2 mg, 0.003 mmol) in dry DMF (1 mL) and DMSO (1 mL) stirred under nitrogen, NHS- PEG685.75 (26 mg, 0.45 mmol) and triethylamine (10 μL, 0.108 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure to obtain a crude oil, which was subjected to preparative HPLC [C18 preparative column: Waters Xterra Prep RP18, 10 μm, 19 × 250 mm. Ambient temperature. Gradient: 10-45% MeCN in 80 minutes. Rf (min) 85] gave BHALys [Lys] ₈ (α-Boc) ₈ (ε-PEG ₅₇₁ ) ₈ as a colorless oil (18 mg, 75%).
LC-MS: (Phobic, TFA) Rf (min) 12.81. ESI (+ ve) m / z = 1246.3 (M / 6), 1068.2 (M / 7), 934.8 (M / 8) 831.2 (M / 9).

ii. BHALys [Lys] ₈ (α-NH ₂ .TFA) ₈ (ε-PEG ₅₇₁ ) ₈
To a stirred solution of BHALys [Lys] ₈ (α-Boc) ₈ (ε-PEG ₅₇₁ ) ₈ (18 mg, 0.002 mmol) in CH ₂ Cl ₂ (3 mL) at 0 ° C., add TFA (45 μL, 0.58 mmol). In addition, stirring at 0 ° C. was continued for 20 minutes, followed by stirring at room temperature for 2 hours. The solvent was removed under reduced pressure to give BHALys [Lys] ₈ (α-NH ₂ .TFA) ₈ (ε-PEG ₅₇₁ ) ₈ as an oil (16 mg, 88%).
LC-MS: (Philic, TFA) Rf (min) 10.36. ESI (+ ve) m / z = 954 (M / 7), 834 (M / 8), 742 (M / 9).
(Example 27)

Production of BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-MTX] ₁₆ Production of the title compound is illustrated with reference to Reaction Scheme 3 (FIG. 17). This reaction scheme is as follows. BHALys [Lys] ₁₆ [α-Fmoc] ₁₆ [ε-Boc] ₁₆ (Structure 2 in FIG. 17; R ₁ = Fmoc, R ₂ = Boc) is reacted with piperidine in DMF to obtain BHALys [Lys] ₁₆ [ α-NH ₂ ] ₁₆ [ε-Boc] ₁₆ (structure 3f; R ₁ = H, R ₂ = Boc) is obtained. Next, BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-Boc] ₁₆ (structure 3g; R ₁ is prepared by reacting structure 3f with an excess amount of PEG ₅₇₀ -NHS in DMF using DIPEA. = PEG ₅₇₀ , R ₂ = Boc). Next, the structure 3g was reacted with TFA (20%) in DCM and then with an ion exchange resin (OH-type) to obtain BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-NH ₂ ] ₁₆ (structure 3h; R ₁ = PEG ₅₇₀ , R ₂ = NH ₂ ) is obtained. The structure 3h was then reacted with excess amounts of α-tBu-γ-MTX-OH, EDC, HOBt and DIPEA in DMSO to produce BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε- (α- tBu-MTX)] ₁₆ (structure 3i; R ₁ = PEG ₅₇₀ , R ₂ = α-tBu-MTX). Next, structure 3i is reacted with TFA to obtain BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-MTX] ₁₆ (structure 3j; R ₁ = PEG ₅₇₀ , R ₂ = MTX).

The use of PEG ₁₇₁₆ , PEG ₂₆₄₅ and PEG ₃₉₇₄ instead of PEG _570- NHS resulted in a large PEG-sized dendrimer construct, which includes structure 3f in DMF and an excess of PEG _MW -CO ₂ H, HOBt And EDC and DIPEA to obtain the structure 3g (R ₁ = PEG _MW , R ₂ = CBz) BHALys [Lys] ₁₆ [α-PEG _MW ] ₁₆ [ε-CBz] ₁₆ is there.
(Example 28)

Production of BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-COCH ₂ CH ₂ CO-Taxol] ₁₆ The production of the title compound is illustrated with reference to Reaction Scheme 5 (FIG. 18). This reaction scheme is as follows. BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-NH ₂ ] ₁₆ (structure 3h; R ₁ = PEG ₅₇₀ , R ₂ = H) and TFA (16 equivalents) in excess of HO ₂ CCH ₂ Reaction with CH ₂ CO-taxol, EDC, HOBt and DIPEA in DMF to produce BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-COCH ₂ CH ₂ CO-taxol] ₁₆ (structure 3l; R ₁ = PEG ₅₇₀ , R ₂ = COCH ₂ CH ₂ CO-taxol).

Use of PEG ₁₇₁₆ , PEG ₂₆₄₅ and PEG ₃₉₇₄ instead of PEG ₅₇₀ in structure 3h yields dendrimer constructs with increased PEG size.
(Example 29)

_{BHALys [Lys] 16 [ε,} ε-PEG 570] Production of ₈ [COCH ₂ CH ₂ CO- Taxol] ₂₄
i. Production of BHALys [Lys] ₈ [α-NH ₂ ] ₈ [ε-CBz] ₈ The production of the intermediate compound is illustrated with reference to Reaction Scheme 6 (Part 1) (FIG. 19A). This reaction scheme is as follows. BHALys [Lys] ₄ [NH ₂ -TFA] ₈ (structure 4; R ₁ = H.TFA) was reacted with excess PNPO-α-Boc-ε-CBz-Lys and DIPEA in DMF to produce BHALys [ Lys] ₈ [α-Boc] ₈ [ε-CBz] ₈ (structure 5a; R ₁ = Boc, R ₂ = CBz) is obtained. After reacting structure 5a with TFA / acetic acid and reacting with an ion exchange resin (0H-type), BHALys [Lys] ₈ [α-NH ₂ ] ₈ [ε-CBz] ₈ (structure 5b; R ₁ = H, R ₂ = CBz).

ii. _{BHALys [Lys] 16 [ε,} ε-PEG 570] illustrates the preparation of ₈ [COCH ₂ CH ₂ CO- taxol] ₂₄ production reaction scheme 6 (Part 2) The title compound with reference to (Figure 19B). This reaction scheme is as follows. BHALys [Lys] ₈ [α-NH ₂ ] ₈ [ε-CBz] ₈ (Structure 5b; R ₁ = H, R ₂ = CBz) and TFA (8 equivalents) are combined with excess DBL-OPNP and DIPEA And DMF in BMFALys [Lys] ₈ [ε-CBz] ₈ [Lys] ₈ [α, α-Boc] ₈ [α, ε-Boc] ₈ (Structure 6a; R ₁ = Boc, R ₂ = CBz). BHALys [Lys] ₈ [ε-NH ₂ ] ₈ [Lys] by reacting structure 6a with H ₂ and Pd (10%) charcoal in acetic acid and then with an ion exchange resin (OH-type) ₈ [α, α-Boc] ₈ [α, ε-Boc] ₈ (structure 6b; R ₁ = Boc, R ₂ = H) is obtained. Reaction of structure 6b with TFA (8 equivalents) in excess of PNPO-α-Boc-ε-CBz-Lys and DIPEA in DMF to yield BHALys [Lys] ₁₆ [ε, ε-CBz] ₈ [Boc ] ₂₄ (Structure 7a; R ₁ = Boc, R ₂ = CBz) is obtained. By reacting structure 7a with H ₂ and Pd (10%) charcoal in acetic acid and then with an ion exchange resin (OH-type), BHALys [Lys] ₁₆ [ε, ε-NH ₂ ] ₈ [ Boc] ₂₄ (structure 7b; R ₁ = Boc, R ₂ = H). By reacting structure 7b with an excess of PEG ₅₇₀ -NHS in DMF using DIPEA, BHALys [Lys] ₁₆ [ε, ε-PEG ₅₇₀ ] ₈ [Boc] ₂₄ (structure 7c; R ₁ = Boc, R ₂ = PEG ₅₇₀ ). Reaction of structure 7c with TFA yields BHALys [Lys] ₁₆ [ε, ε-PEG ₅₇₀ ] ₈ [NH ₂ -TFA] ₂₄ (structure 7d; R ₁ = H.TFA, R ₂ = PEG ₅₇₀ ). The structure 7dI is reacted with excess HO ₂ CCH ₂ CH ₂ CO-taxol, EDC, HOBt and DIPEA in DMF to give BHALys [Lys] ₁₆ [ε, ε-PEG ₅₇₀ ] ₈ [COCH ₂ CH ₂ CO— Taxol] ₂₄ (structure 7e; R ₁ = COCH ₂ CH ₂ CO-taxol, R ₂ = PEG ₅₇₀ ).

The use of PEG ₁₇₁₆ , PEG ₂₆₄₅ and PEG ₃₉₇₄ instead of PEG _570- NHS resulted in a large PEG-sized dendrimer construct, which included structure 7b in excess of PEG _MW -CO ₂ H and HOBt in DMF. And reaction using EDC and DIPEA to obtain BHALys [Lys] ₁₆ [ε, ε-PEG _MW ] ₈ [Boc] ₂₄ (structure 7c; R ₁ = Boc, R ₂ = PEG _MW ) Conditions are needed.
(Example 30)

Production of well-defined PEG moieties: PEG ₁₇₁₆ , PEG ₂₆₄₅ , PEG ₃₉₇₄ The production of the above alternative PEG moiety to substitute in the above reaction scheme is illustrated using Reaction Scheme 7 (FIG. 20). This reaction scheme is as follows. i. HO ₂ C-PEG ₁₁₄₆ -NH ₂ is reacted with PEG ₅₇₀ -NHS in DMF-buffer pH 8.5 to give PEG ₁₇₁₆ -CO ₂ H. ii. PEG ₁₇₁₆ -CO ₂ H is reacted with DCC and NHS and then reacted with HO ₂ C-PEG ₁₁₄₆ -NH ₂ in DMF-buffer pH 8.5 to give PEG ₂₈₄₅ -CO ₂ H. iii. PEG ₂₈₄₅ -CO ₂ H is reacted with DCC and NHS and then reacted with HO ₂ C-PEG ₁₁₄₆ -NH ₂ in DMF-buffer pH 8.5 to obtain PEG ₃₉₇₄ -CO ₂ H.
(Example 31)

DCM（200mL）中の3-ブロモプロピルアミン臭化水素酸塩（10.0g、45.6mmol）とN-(ベンジルオキシカルボニルオキシ)-スクシンイミド（11.22g、47.9mmol）の氷冷混合物に、TEA（6.91g、68.5mmol）を滴下した。撹拌した混合物を終夜、室温まで温まらせた後、水（3回）、ブラインで洗浄し、乾燥（MgSO₄）し、濾過し、濃縮することにより、11.43g（92％）のN-(ベンジルオキシカルボニル)-3-ブロモプロピルアミンを淡黄色油状物として得た。 _{BHALys [Lys] 2 [Su (} NPN) 2] 4 [α-tBu-MTX] 4 production of [PEG _{570] 4}
i. Production of N- (benzyloxycarbonyl) -3-bromopropylamine

To an ice-cooled mixture of 3-bromopropylamine hydrobromide (10.0 g, 45.6 mmol) and N- (benzyloxycarbonyloxy) -succinimide (11.22 g, 47.9 mmol) in DCM (200 mL) was added TEA (6.91 g, 68.5 mmol) was added dropwise. The stirred mixture was allowed to warm to room temperature overnight, then washed with water (3 times), brine, dried (MgSO ₄ ), filtered and concentrated to give 11.43 g (92%) of N- (benzyl Oxycarbonyl) -3-bromopropylamine was obtained as a pale yellow oil.

DMF（150mL）中のN-(ベンジルオキシカルボニル)-3-ブロモプロピルアミン（11.20g、41.2mmol）とN-BOCジアミノプロパン（7.16g、41.2mmol）の混合物を室温で撹拌したものに、TEA（17.1mL、123.5mmol）を滴下した。その混合物を70℃に1時間加熱した後、溶媒の約2/3を減圧下で除去した。次に、濃縮したDMF混合物を水（400mL）で希釈し、エーテル（200mL×3）で洗浄することにより、過剰アルキル化副生成物の大半を除去した。次に、そのDMF/水性混合物を塩基性にし（1.0M NaOH）、エーテル（200mL×5）で抽出した。次に、合わせたエーテル抽出物を、未反応のN-BOCジアミノプロパンを除去するために水（200mL×3）で洗浄し、乾燥し（MgSO₄）、濾過し、濃縮することにより、7.13g（47％）の[BOC][Cbz][NPN]₂を、無色透明な油状物として得た。 ii. Manufacture of [BOC] [Cbz] [NPN] ₂

To a stirred mixture of N- (benzyloxycarbonyl) -3-bromopropylamine (11.20 g, 41.2 mmol) and N-BOC diaminopropane (7.16 g, 41.2 mmol) in DMF (150 mL) at room temperature was added TEA. (17.1 mL, 123.5 mmol) was added dropwise. The mixture was heated to 70 ° C. for 1 hour, after which about 2/3 of the solvent was removed under reduced pressure. The concentrated DMF mixture was then diluted with water (400 mL) and washed with ether (200 mL × 3) to remove most of the excess alkylated byproduct. The DMF / aqueous mixture was then basified (1.0 M NaOH) and extracted with ether (200 mL × 5). The combined ether extracts were then washed with water (200 mL × 3) to remove unreacted N-BOC diaminopropane, dried (MgSO ₄ ), filtered and concentrated to give 7.13 g (47%) of [BOC] [Cbz] [NPN] ₂ was obtained as a clear colorless oil.

トルエン（60mL）中の[BOC][Cbz][NPN]₂（6.55g、17.9mmol）の混合物を室温で撹拌したものに、無水コハク酸（1.79g、17.9mmol）を加えた。その混合物を70℃に1時間加熱してから濃縮した。次に残渣をEA/エーテル（5：1）に溶解し、NaOH（1.0M、100mL×2）で洗浄した。次に塩基洗浄液をエーテルで洗浄してから、中和（HCl、1.0M、100mL×2）した。次に、その水性混合物をEA（250mL×3）で洗浄し、乾燥し（MgSO₄）、濾過し、濃縮することにより、6.97g（84％）の[BOC][Cbz][NPN]₂SuOHを無色の粘稠な油状物として得た。 iii. [BOC] [Cbz] [NPN] ₂ Production of SuOH

To a stirred mixture of [BOC] [Cbz] [NPN] ₂ (6.55 g, 17.9 mmol) in toluene (60 mL) at room temperature was added succinic anhydride (1.79 g, 17.9 mmol). The mixture was heated to 70 ° C. for 1 hour and then concentrated. The residue was then dissolved in EA / ether (5: 1) and washed with NaOH (1.0 M, 100 mL × 2). Next, the base washing solution was washed with ether and then neutralized (HCl, 1.0 M, 100 mL × 2). The aqueous mixture was then washed with EA (250 mL × 3), dried (MgSO ₄ ), filtered and concentrated to give 6.97 g (84%) of [BOC] [Cbz] [NPN] ₂ SuOH Was obtained as a colorless viscous oil.

EA（150mL）中の4-ニトロフェノール（1.91g、13.7mmol）と[BOC][Cbz][NPN]₂SuOH（6.39g、13.7mmol）の混合物を室温で撹拌したものに、EA（50mL）に溶解したDCC（2.97g、14.4mmol）を加えた。その混合物を室温で終夜、撹拌放置してから、濾過した（DCUを除去するため）。次に、その混合物をK₂CO₃（1.0M）/ブライン 1：1（300mL×3）、ブラインで洗浄し、乾燥（MgSO₄）し、濾過し、濃縮することにより、[BOC][Cbz][NPN]₂SuOPNP（7.80g）を粗物質として得た。 iv. [BOC] [Cbz] [NPN] ₂ Manufacture of SuOPNP

A mixture of 4-nitrophenol (1.91 g, 13.7 mmol) and [BOC] [Cbz] [NPN] ₂ SuOH (6.39 g, 13.7 mmol) in EA (150 mL) was stirred at room temperature with EA (50 mL) DCC (2.97 g, 14.4 mmol) dissolved in was added. The mixture was left stirring at room temperature overnight and then filtered (to remove DCU). The mixture was then washed with K ₂ CO ₃ (1.0 M) / brine 1: 1 (300 mL × 3), brine, dried (MgSO ₄ ), filtered and concentrated to give [BOC] [Cbz ] [NPN] ₂ SuOPNP (7.80 g) was obtained as a crude material.

撹拌したEtOH（100mL）中の[BOC][Cbz][NPN]₂SuOPNP（1.16g、1.98mmol）とTEA（2.0mL、14.4mmol）の混合物を70℃で2日間加熱し、濃縮してから、酢酸エチル（120mL）に取り出した。次にその混合物をK₂CO₃溶液（5％、200mL×4）、ブラインで洗浄し、乾燥（MgSO₄）し、濾過し、濃縮することにより、0.87g（90％）の[BOC][Cbz][NPN]₂SuOEtを無色の粘性油状物として得た。
LCMS（LC：親水性（philic）, ホルメート、RT＝9.3分；MS（M(計算値) C₂₅H₃₉N₃O₇＝493.61）：511 ([M+NH₄]⁺, 13％)、494([M+H]⁺, 100％)、438 ([M-t-Bu+H]⁺, 15％)、394 ([M-BOC+H]⁺, 13％）。
¹H (CDCl₃)：δ 7.30-7.38 (m, 5H), 5.70（br s, 0.5H), 5.25 (br s, 0.5H), 5.10（br s, 0.5H), 5.10, 5.08 (2s, 2H), 4.70（br s, 0.5H), 4.12 (q, J＝9.0Hz, 2H), 2.95-3.45 (m, 8H), 2.52-2.70（m, 4H), 1.73-1.90（m, 2H), 1.60-1.70（m, 2H), 1.42, 1.44 (2s, 9H), 1.25 (t, J＝9.0Hz, 3H)。 v. [BOC] [Cbz] [NPN] ₂ Manufacture of SuOEt

A stirred mixture of [BOC] [Cbz] [NPN] ₂ SuOPNP (1.16 g, 1.98 mmol) and TEA (2.0 mL, 14.4 mmol) in EtOH (100 mL) was heated at 70 ° C. for 2 days and concentrated. Into ethyl acetate (120 mL). The mixture was then washed with K ₂ CO ₃ solution (5%, 200 mL × 4), brine, dried (MgSO ₄ ), filtered and concentrated to give 0.87 g (90%) [BOC] [ Cbz] [NPN] ₂ SuOEt was obtained as a colorless viscous oil.
LCMS (LC: hydrophilic, formate, RT = 9.3 min; MS (M (calculated) C ₂₅ H ₃₉ N ₃ O ₇ = 493.61): 511 ([M + NH ₄ ] ⁺ , 13%), 494 ([M + H] ⁺ , 100%), 438 ([Mt-Bu + H] ⁺ , 15%), 394 ([M-BOC + H] ⁺ , 13%).
¹ H (CDCl ₃ ): δ 7.30-7.38 (m, 5H), 5.70 (br s, 0.5H), 5.25 (br s, 0.5H), 5.10 (br s, 0.5H), 5.10, 5.08 (2s, 2H), 4.70 (br s, 0.5H), 4.12 (q, J = 9.0Hz, 2H), 2.95-3.45 (m, 8H), 2.52-2.70 (m, 4H), 1.73-1.90 (m, 2H) , 1.60-1.70 (m, 2H), 1.42, 1.44 (2s, 9H), 1.25 (t, J = 9.0Hz, 3H).

DMF/H₂O（9：1、20mL）中の[BOC][Cbz][NPN]₂SuOEt（0.88g、1.77mmol）の混合物を撹拌したものに、ギ酸アンモニウム（224mg、3.55mmol）およびPd/C（10％、470mg）を加えた。その混合物を室温で2時間撹拌してから濾過（0.2μm PALLフィルターディスク）し、濃縮した。残渣を水にとり、濃縮した（2回）。次にこれをMeOHおよびDCMで繰り返すことにより、0.54g（84％）の[BOC][NH₂][NPN]₂SuOEtを無色透明な油状物として得た。
LCMS（LC：親水性（philic）, TFA、RT＝6.2分；MS（M(計算値) C₁₇H₃₃N₃O₅＝359.47）：360 ([M+H]⁺, 100％）。
¹H (CDCl₃)：δ 5.30（br s, 1H), 4.80（br s, 1H), 4.12 (q, J＝9.0Hz, 2H), 3.29-3.46 (m, 4H), 3.14 (m, 1H), 3.07 (m, 1H), 2.56-2.80（m, 6H), 1.60-1.90（m, 4H), 1.42, 1.43 (2s, 9H), 1.25 (t, J＝9.0Hz, 3H)。
vii．[BOC][PEG ₅₇₀ ][NPN] ₂ SuOEtの製造

DCM（2mL）中の[BOC][NH₂][NPN]₂SuOEt（157mg、0.44mmol）の混合物を撹拌したものに、TEA（121μl、0.87mmol）およびPEG₅₇₀-NHS（300mg、0.44mmol）をDCM（2mL）溶液として加えた。その混合物を室温で終夜撹拌してから、fcc（2-10％ MeOH/DCM）で精製することにより、331mg（82％）の[BOC][PEG₅₇₀][NPN]₂SuOEtを無色透明の油状物として得た。
LCMS（LC：親水性（philic）, TFA、RT＝8.2分；MS（M(計算値) C₄₃H₈₃N₃O₁₈＝930.15）：948 ([M+NH₄]⁺, 12％)、931 ([M+H]⁺, 2％)、416 (1/2[M-BOC+2H⁺], 100％）。
¹H (CDCl₃)：δ 7.10（br s, 1H), 7.03 (br s, 1H), 4.16 (q, J＝9.0Hz, 2H), 3.72 (m, 2H), 3.58-3.66 (m, 36H), 3.52-3.56 (m, 2H), 3.47 (s, 2H), 2.96-3.42 (m, 7H), 3.37 (s, 4H), 2.70（s, 4H), 2.60（m, 4H), 2.47 (m, 2H), 1.60-1.90（m, 4H), 1.41, 1.43 (2s, 9H), 1.24 (t, J＝9.0Hz, 3H)。 vi. [BOC] [NH ₂ ] [NPN] ₂ Manufacture of SuOEt

To a stirred mixture of [BOC] [Cbz] [NPN] ₂ SuOEt (0.88 g, 1.77 mmol) in DMF / H ₂ O (9: 1, 20 mL) was added ammonium formate (224 mg, 3.55 mmol) and Pd. / C (10%, 470 mg) was added. The mixture was stirred at room temperature for 2 hours, then filtered (0.2 μm PALL filter disk) and concentrated. The residue was taken up in water and concentrated (twice). This was then repeated with MeOH and DCM to give 0.54 g (84%) of [BOC] [NH ₂ ] [NPN] ₂ SuOEt as a clear colorless oil.
LCMS (LC: hydrophilic, TFA, RT = 6.2 min; MS (M (calculated) C ₁₇ H ₃₃ N ₃ O ₅ = 359.47): 360 ([M + H] ⁺ , 100%).
¹ H (CDCl ₃ ): δ 5.30 (br s, 1H), 4.80 (br s, 1H), 4.12 (q, J = 9.0Hz, 2H), 3.29-3.46 (m, 4H), 3.14 (m, 1H ), 3.07 (m, 1H), 2.56-2.80 (m, 6H), 1.60-1.90 (m, 4H), 1.42, 1.43 (2s, 9H), 1.25 (t, J = 9.0Hz, 3H).
vii. [BOC] [PEG ₅₇₀ ] [NPN] ₂ Manufacture of SuOEt

To a stirred mixture of [BOC] [NH ₂ ] [NPN] ₂ SuOEt (157 mg, 0.44 mmol) in DCM (2 mL) was added TEA (121 μl, 0.87 mmol) and PEG ₅₇₀ -NHS (300 mg, 0.44 mmol). Was added as a DCM (2 mL) solution. The mixture was stirred at room temperature overnight and then purified by fcc (2-10% MeOH / DCM) to obtain 331 mg (82%) of [BOC] [PEG ₅₇₀ ] [NPN] ₂ SuOEt as a clear, colorless oil Obtained as a thing.
LCMS (LC: hydrophilic, TFA, RT = 8.2 min; MS (M (calculated) C ₄₃ H ₈₃ N ₃ O ₁₈ = 930.15): 948 ([M + NH ₄ ] ⁺ , 12%), 931 ([M + H] ⁺ , 2%), 416 (1/2 [M-BOC + 2H ⁺ ], 100%).
¹ H (CDCl ₃ ): δ 7.10 (br s, 1H), 7.03 (br s, 1H), 4.16 (q, J = 9.0Hz, 2H), 3.72 (m, 2H), 3.58-3.66 (m, 36H ), 3.52-3.56 (m, 2H), 3.47 (s, 2H), 2.96-3.42 (m, 7H), 3.37 (s, 4H), 2.70 (s, 4H), 2.60 (m, 4H), 2.47 ( m, 2H), 1.60-1.90 (m, 4H), 1.41, 1.43 (2s, 9H), 1.24 (t, J = 9.0 Hz, 3H).

DCM（2mL）中の[BOC][PEG₅₇₀][NPN]₂SuOEt（180mg、0.19mmol）の混合物を撹拌したものにTFA（0.50mL）を加えた。その混合物を室温で6時間撹拌し、濃縮し、H₂Oを加え、濃縮した（2回）。次に残渣を再びH₂O（20mL）にとり、濾過（0.2μm PALLフィルターディスク）してから、凍結乾燥することにより、0.17g（93％）の[NH₂.TFA][PEG₅₇₀][NPN]₂SuOEt を無色透明の油状物として得た。
LCMS（LC：親水性（philic）, TFA、RT＝5.9分；MS（M(計算値) C₃₈H₇₅N₃O₁₆＝830.03)：831 ([M+H]⁺, 7％)、425 (1/2[M+Na⁺+H⁺], 30％)、416 (1/2[M+2H⁺], 100％）。
¹H (D₂O)：δ 4.17 (q, J＝9.0Hz, 2H), 3.80（t, J＝6.0Hz, 2H), 3.62-3.75 (m, 43H), 3.38-3.53 (m, 4H), 3.40（s, 3H), 2.90-3.31 (m, 4H), 2.77 (s, 4H), 2.64-2.89 (m, 4H), 2.52-2.58 (m, 2H), 1.72-2.11 (m, 4H), 1.13 (t, J＝9.0Hz, 3H)。 viii. [NH ₂ -TFA] [PEG ₅₇₀ ] [NPN] ₂ Manufacture of SuOEt

To a stirred mixture of [BOC] [PEG ₅₇₀ ] [NPN] ₂ SuOEt (180 mg, 0.19 mmol) in DCM (2 mL) was added TFA (0.50 mL). The mixture was stirred at room temperature for 6 hours, concentrated, H ₂ O was added and concentrated (twice). The residue is then taken up again in H ₂ O (20 mL), filtered (0.2 μm PALL filter disc) and lyophilized to give 0.17 g (93%) of [NH ₂ .TFA] [PEG ₅₇₀ ] [NPN ] ₂ SuOEt was obtained as a colorless and transparent oil.
LCMS (LC: hydrophilic, TFA, RT = 5.9 min; MS (calculated) C ₃₈ H ₇₅ N ₃ O ₁₆ = 830.03): 831 ([M + H] ⁺ , 7%), 425 (1/2 [M + Na ⁺⁺ H ⁺ ], 30%), 416 (1/2 [M + 2H ⁺ ], 100%).
¹ H (D ₂ O): δ 4.17 (q, J = 9.0Hz, 2H), 3.80 (t, J = 6.0Hz, 2H), 3.62-3.75 (m, 43H), 3.38-3.53 (m, 4H) , 3.40 (s, 3H), 2.90-3.31 (m, 4H), 2.77 (s, 4H), 2.64-2.89 (m, 4H), 2.52-2.58 (m, 2H), 1.72-2.11 (m, 4H) , 1.13 (t, J = 9.0Hz, 3H).

DMF（0.5mL）中の[NH₂.TFA][PEG₅₇₀][NPN]₂SuOEt（30mg、31.7μmol）とα-tBu-γ-MTX-OH（16.2mg、31.7μmol）｛C.L. Francis, Q. Yang、N.K. Hart, F. Widmer, M.K. MantheyおよびH. Ming He-Williams, Aust. J. Chem. 2002, 55, 635｝の混合物を0℃で撹拌したものに、PyBOP（18mg、34.8μmol）およびDIPEA（23μL、0.127mmol）を加えた。その混合物を0℃で30分間撹拌してから、室温で3時間撹拌した。DMFを除去し、残渣をPTLC（7％MeOH, 93％DCM、Rf＝0.3）で精製することにより、23mg（55％）の[α-tBu-MTX][PEG₁₅₇₀][NPN]₂SuOEtを橙色油状物として得た。
LCMS（LC：親水性（philic）, TFA、RT＝8.0分；MS（M(計算値) C₆₂H₁₀₃N₁₁O₂₀＝1322.57)：1323 ([M+H]⁺, 2％)、662 (1/2[M+2H⁺], 17％)、634 (1/2[M-tBu+2H⁺], 82％)。 ix. [α-tBu-MTX] [PEG ₁₅₇₀ ] [NPN] ₂ Production of SuOEt

[NH ₂ .TFA] [PEG ₅₇₀ ] [NPN] ₂ SuOEt (30 mg, 31.7 μmol) and α-tBu-γ-MTX-OH (16.2 mg, 31.7 μmol) in DMF (0.5 mL) {CL Francis, Q A mixture of Yang, NK Hart, F. Widmer, MK Manthey and H. Ming He-Williams, Aust. J. Chem. 2002, 55, 635} was stirred at 0 ° C. with PyBOP (18 mg, 34.8 μmol). And DIPEA (23 μL, 0.127 mmol) were added. The mixture was stirred at 0 ° C. for 30 minutes and then at room temperature for 3 hours. DMF was removed and the residue was purified by PTLC (7% MeOH, 93% DCM, Rf = 0.3) to give 23 mg (55%) [α-tBu-MTX] [PEG ₁₅₇₀ ] [NPN] ₂ SuOEt Obtained as an orange oil.
LCMS (LC: hydrophilic, TFA, RT = 8.0 min; MS (M (calculated) C ₆₂ H ₁₀₃ N ₁₁ O ₂₀ = 1322.57): 1323 ([M + H] ⁺ , 2%), 662 (1/2 [M + 2H ⁺ ], 17%), 634 (1/2 [M-tBu + 2H ⁺ ], 82%).

THF/H₂O（2：1、9mL）中の[α-tBu-MTX][PEG₁₅₇₀][NPN]₂SuOEt（109mg、82.4μmol）の混合物を撹拌したものに、NaOH（0.16mL、1.0M）を加えた。反応を室温で16時間撹拌放置し、必要であればNaOHを追加した（反応をtlcで判定）。反応が完了した後、pHをHCl（1.0M）で中性に調節した。次に溶媒を除去し、残渣をMeOHにとり、塩を除去するために濾過した。次に、残渣をPTLC（18％MeOH, 82％DCM、Rf＝0.4）で精製することにより、52mg（49％）の[α-tBu-MTX][PEG₅₇₀][NPN]₂SuOHを橙色油状物として得た。
LCMS（LC：親水性（philic）, TFA、RT＝6.8分；MS（M(計算値) C₆₀H₉₉N₁₁O_2O＝1294.52)：1317 ([M+Na]⁺, 3％)、1295 ([M+H]⁺, 2％)、648 (1/2[M+2H⁺], 10％)、620 (1/2[M-tBu+2H⁺], 74％)、419 (100％）。 x. [α-tBu-MTX] [PEG ₅₇₀ ] [NPN] ₂ Production of SuOH

To a stirred mixture of [α-tBu-MTX] [PEG ₁₅₇₀ ] [NPN] ₂ SuOEt (109 mg, 82.4 μmol) in THF / H ₂ O (2: 1, 9 mL) was added NaOH (0.16 mL, 1.0 M) was added. The reaction was left to stir at room temperature for 16 hours and NaOH was added if necessary (reaction determined by tlc). After the reaction was complete, the pH was adjusted to neutral with HCl (1.0 M). The solvent was then removed and the residue was taken up in MeOH and filtered to remove the salt. The residue was then purified by PTLC (18% MeOH, 82% DCM, Rf = 0.4) to give 52 mg (49%) of [α-tBu-MTX] [PEG ₅₇₀ ] [NPN] ₂ SuOH as an orange oil Obtained as a thing.
LCMS (LC: hydrophilic, TFA, RT = 6.8 min; MS (M (calculated) C ₆₀ H ₉₉ N ₁₁ O _2O = 1294.52): 1317 ([M + Na] ⁺ , 3%), 1295 ([M + H] ⁺ , 2%), 648 (1/2 [M + 2H ⁺ ], 10%), 620 (1/2 [M-tBu + 2H ⁺ ], 74%), 419 (100% ).

xi. _{BHALys [Lys] 2 [Su (} NPN) 2] 4 [α-tBu-MTX] 4 production of [PEG _{570] 4}
[Α-tBu-MTX] [PEG ₅₇₀ ] [NPN] ₂ SuOH (10 mg, 7.7 μmol) and BHALys [Lys] ₂ [NH ₂ .TFA] ₄ (1.43 mg, 1.4 μmol) in DMF (1.2 mL) To the stirred mixture at 0 ° C., PyBOP (4.0 mg, 7.7 μmol) and DIPEA (3.9 μL, 22.4 μmol) were added. The mixture was stirred at 0 ° C. for 30 minutes and then stirred at room temperature for 3 hours. DMF was removed and the residue was purified by preparative HPLC (Waters Xterra MS C ₁₈ , 10 μm, 19 × 250 mm, 30-60% ACN, 0.1% TFA, 8 mL / min, RT = 34 min) to give 2 mg ( BHALys [Lys] ₂ [Su (NPN) ₂ ] ₄ [α-tBu-MTX] ₄ [PEG ₅₇₀ ] ₄ of 25% {mostly eluted in voids] was obtained.
LCMS (LC: hydrophilic, TFA, RT = 8.0 min; MS: 1136 (1/5 [M + 5H ⁺ ], 18%), 946 (1/6 [M + 6H ⁺ ], 100%) 812 (1/7 [M + 7H ⁺ ], 22%) Conversion value 5,673.34 (M (calculated value) C ₂₇₁ H ₄₃₇ N ₅₁ O ₇₉ = 5,673.80).
(Example 32)

BHALys [Lys] ₄ [Su (NPN) ₂ ] ₈ [α-tBu-MTX] ₈ [PEG ₅₇₀ ] ₈ Production of BHALys [Lys] ₄ [NH ₂ using the same method as described in Example 31 .TFA] ₈ reacts with [α-tBu-MTX] [PEG ₅₇₀ ] [NPN] ₂ SuOH to produce BHALys [Lys] ₄ [Su (NPN) ₂ ] ₈ [α-tBu-MTX] ₈ [PEG ₅₇₀ ] ₈ (10 mg) (51%). Preparative HPLC (5-60% ACN, 90 minutes, RT 54 minutes).
LCMS (LC: hydrophilic, TFA, RT = 9.0 min; MS: 1614 (1/7 [M + 7H ⁺ ], 26%), 1413 (1/8 [M + 8H ⁺ ], 73%) , 1256 (1/9 [M + 9H ⁺ ], 100%) Conversion value 11,294.54 (M (calculated value) C ₅₃₅ H ₈₇₃ N ₁₀₃ O ₁₅₉ = 11,292.52).
(Example 33)

BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [α-tBu-MTX] ₁₆ [PEG ₅₇₀ ] ₁₆ Production of BHALys [Lys] ₈ [NH ₂ using the same method as described in Example 31 .TFA] ₁₆ is reacted with [α-tBu-MTX] [PEG ₅₇₀ ] [NPN] ₂ SuOH to produce BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [α-tBu-MTX] ₁₆ [PEG ₅₇₀ ] ₁₆ (6 mg) (46%). Preparative HPLC (5-60% ACN, 90 minutes, RT66 minutes).
LCMS (LC: hydrophilic, TFA, RT = 9.0 min; MS: 2254 (1/10 [M + 10H ⁺ ], 24%), 2049 (1/11 [M + 11H ⁺ ], 56%) , 1879 (1/12 [M + 12H ⁺ ], 100%), 1734 (1/13 [M + 13H ⁺ ], 55%), converted value 22,531.91 (M (calculated value) C ₁₀₆₃ H ₁₇₄₅ N ₂₀₇ O ₃₁₉ = 22,529.73).

BHALys [ ³ H-Lys] ₈ [Su (NPN) ₂ ] ₁₆ [α-tBu-MTX] ₁₆ [PEG ₅₇₀ ] ₁₆ was produced by the same method. 15 mg (65%). 1.27mCi / g.
(Example 34)

BHALys [Lys] ₁₆ [Su (NPN) ₂ ] ₃₂ [α-tBu-MTX] ₃₂ [PEG ₅₇₀ ] ₃₂
By reacting BHALys [Lys] ₁₆ [NH ₂ .TFA] ₃₂ with [α-tBu-MTX] [PEG ₅₇₀ ] [NPN] ₂ SuOH using a procedure similar to that described in Example 31, BHALys [Lys] ₁₆ [Su (NPN) ₂ ] ₃₂ [α-tBu-MTX] ₃₂ [PEG ₅₇₀ ] ₃₂ (7 mg) (44%) was obtained. Preparative HPLC (5-60% ACN, 90 minutes, RT71 minutes). LCMS (LC: hydrophilic, TFA, RT = 9.2 min; MS: (M (calculated) C ₂₁₁₉ H ₃₄₈₉ N ₄₁₅ O ₆₃₉ = 45,004.27).
(Example 35)

DMF（2mL）中の[BOC][NH₂][NPN]₂SuOEtの混合物を撹拌したものに、TEA（2等量）およびPEG₁₁₀₀NHS（1等量）をDMF/DCM（2mL）溶液として加えた。その混合物を室温で終夜撹拌し、濃縮した後、fccで精製することにより、[BOC][PEG₁₁₀₀][NPN]₂SuOEtを無色透明の油状物として得た。 i. [BOC] [PEG ₁₁₀₀ ] [NPN] ₂ Manufacture of SuOEt

To a stirred mixture of [BOC] [NH ₂ ] [NPN] ₂ SuOEt in DMF (2 mL), TEA (2 eq) and PEG ₁₁₀₀ NHS (1 eq) as a DMF / DCM (2 mL) solution added. The mixture was stirred at room temperature overnight, concentrated, and purified by fcc to give [BOC] [PEG ₁₁₀₀ ] [NPN] ₂ SuOEt as a colorless transparent oil.

DCM（2mL）中の[BOC][PEG₁₁₀₀][NPN]₂SuOEt（180mg）の混合物を撹拌したものに、TFA（0.50mL）を加えた。その混合物を室温で6時間撹拌し、濃縮し、H₂Oを加え、濃縮した（2回）。次に残渣を再びH₂O（20mL）にとり、濾過（0.2μm PALLフィルターディスク）してから凍結乾燥することにより、[NH₂.TFA][PEG₁₁₀₀][NPN]₂SuOEtを無色透明の油状物として得た。 ii. [NH ₂ .TFA] [PEG ₁₁₀₀ ] [NPN] ₂ Manufacture of SuOEt

To a stirred mixture of [BOC] [PEG ₁₁₀₀ ] [NPN] ₂ SuOEt (180 mg) in DCM (2 mL) was added TFA (0.50 mL). The mixture was stirred at room temperature for 6 hours, concentrated, H ₂ O was added and concentrated (twice). The residue is then taken up again in H ₂ O (20 mL), filtered (0.2 μm PALL filter disc) and lyophilized to give [NH ₂ .TFA] [PEG ₁₁₀₀ ] [NPN] ₂ SuOEt as a clear, colorless oil Obtained as a thing.

DMF（0.5mL）中の[NH₂.TFA][PEG₁₁₀₀][NPN]₂SuOEtとα-tBu-γ-MTX-OH（1等量）｛C.L. Francis, Q. Yang, N.K. Hart, F. Widmer, M.K. MantheyおよびH. Ming He-Williams, Aust. J. Chem. 2002, 55, 635｝の混合物を0℃で撹拌したものに、PyBOP（1.2等量）およびDIPEA（3等量）を加えた。その混合物を0℃で30分間撹拌した後、室温で3時間撹拌した。DMFを除去し、残渣をPTLCで精製することにより、[α-tBu-MTX][PEG₁₁₀₀][NPN]₂SuOEtを橙色油状物として得た。 iii. [α-tBu-MTX] [PEG ₁₁₀₀ ] [NPN] ₂ SuOEt

[NH ₂ .TFA] [PEG ₁₁₀₀ ] [NPN] ₂ SuOEt and α-tBu-γ-MTX-OH (1 equivalent) in DMF (0.5 mL) {CL Francis, Q. Yang, NK Hart, F. PyBOP (1.2 eq) and DIPEA (3 eq) were added to a mixture of Widmer, MK Manthey and H. Ming He-Williams, Aust. J. Chem. 2002, 55, 635} stirred at 0 ° C. It was. The mixture was stirred at 0 ° C. for 30 minutes and then stirred at room temperature for 3 hours. DMF was removed and the residue was purified by PTLC to give [α-tBu-MTX] [PEG ₁₁₀₀ ] [NPN] ₂ SuOEt as an orange oil.

THF/H₂O（2：1、9mL）中の[α-tBu-MTX][PEG1₁₁₀₀][NPN]₂SuOEtの混合物を撹拌したものに、NaOH（2等量、1.0M）を加えた。反応を室温で16時間、撹拌放置し、必要であればNaOHを追加した（反応をtlcで判定）。反応が完了した後、pHをHCl（1.0M）で中性に調節した。次に溶媒を除去し、残渣をMeOHにとり、塩を除去するために濾過した。次に、残渣をPTLCで精製することにより、[α-tBu-MTX][PEG₁₁₀₀][NPN]₂SuOHを橙色油状物として得た。 iv. Production of [α-tBu-MTX] [PEG ₁₁₀₀ ] [NPN] ₂ SuOH

To a stirred mixture of [α-tBu-MTX] [PEG1 ₁₁₀₀ ] [NPN] ₂ SuOEt in THF / H ₂ O (2: 1, 9 mL) was added NaOH (2 eq, 1.0 M). . The reaction was left to stir at room temperature for 16 hours and NaOH was added if necessary (reaction determined by tlc). After the reaction was complete, the pH was adjusted to neutral with HCl (1.0 M). The solvent was then removed and the residue was taken up in MeOH and filtered to remove the salt. The residue was then purified by PTLC to give [α-tBu-MTX] [PEG ₁₁₀₀ ] [NPN] ₂ SuOH as an orange oil.

v. BHALys [Lys] ₈ preparation of _{[Su (NPN) 2] 16} [α-tBu-MTX] 16 [PEG 1100] 16
A mixture of [α-tBu-MTX] [PEG ₁₁₀₀ ] [NPN] ₂ SuOH (1.1 equivalents per NH ₂ ) and BHALys [Lys] ₈ [NH ₂ .TFA] ₁₆ in DMF (1.2 mL) at 0 ° C. To the stirred one was added PyBOP (1.2 equivalents per NH ₂ ) and DIPEA (3 equivalents per NH ₂ ). The mixture was stirred at 0 ° C. for 30 minutes and then at room temperature for 3 hours. DMF was removed, and the residue was purified by preparative HPLC (Waters Xterra MS C ₁₈ , 10 μm, 19 × 250 mm) to obtain BHALys [Lys] ₈ [Su (NPN) ₂ ] ₁₆ [α-tBu-MTX] ₁₆ [PEG ₁₁₀₀ ] ₁₆ was obtained.
(Example 36)

Production of HOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz] This synthesis is illustrated in FIG.
i. Preparation of MeOGlyLys [α-Boc] [ε-CBz] Methyl glycinate hydrochloride (12.56 g, 0.110 mol) suspended in a mixture of triethylamine (30.36 g, 0.300 mol) and dimethylformamide (200 ml) was stirred. PNPO-α-Boc-ε-CBz-Lys (50.15 g, 0.100 mol) was added. After stirring at ambient temperature for 16 hours, the volatile components were evaporated under reduced pressure and the residue was partitioned between ethyl acetate (200 ml) and 5% aqueous sodium carbonate (175 ml). The aqueous phase is discarded and the ethyl acetate phase is washed with a fresh 5% aqueous sodium carbonate solution (200 ml × 4), then with 0.25 M hydrochloric acid (50 ml × 2) and then with a saturated aqueous sodium chloride solution (50 ml). Washed. The ethyl acetate solution was dried (magnesium sulfate), filtered, and the solvent was evaporated under reduced pressure to give MeOGlyLys [α-Boc] [ε-CBz] (44.39 g, 98%) as a colorless oil. .
¹ H nmr (300 MHz, CD ₃ OD) δ (ppm): 1.3-1.8 (m, 6H); 1.44 (s, 9H); 3.13 (t, J 6.6 Hz, 2H); 3.70 (s, 3H); 3.88 (d, J 17.7Hz, 1H); 3.99 (d, J 17.7Hz, 1H); 4.04 (m, 1H); 5.06 (s, 2H); 7.2-7.4 (m, 5H).
LC / MS (hydrophobicity / formate): ESI (+ ve) Found ^{[M + H] + m /} z = 452.0; ( as _{C 22 H 34 N 3 O 7} ) Calculated 452.2. Rf (minutes) = 5.2.

ii. Production of MeOGlyLys [α-NH ₂ .TFA] [ε-CBz]
MeOGlyLys [α-Boc] [ε-CBz] (43.36 g, 96.0 mmol) was dissolved in acetic acid (150 ml) and the solution was stirred at ice bath temperature until the acetic acid began to freeze. The ice bath was removed and trifluoroacetic acid was added slowly with stirring until the acetic acid crystals dissolved. The solution was once again placed in an ice bath and the remainder of the trifluoroacetic acid (total 150 ml) was added slowly. Freezing of acetic acid never occurred again. After removing the ice bath and stirring the solution at ambient temperature for 5 hours, the volatile components were evaporated as completely as possible under reduced pressure. The remaining viscous oil was dissolved in methanol (200 ml) and again subjected to a rotary evaporator under reduced pressure to give an oil. This process was repeated five more times with 200 ml of methanol each, and then residual methanol was removed as much as possible under a reduced pressure of 0.1 torr. The product MeOGlyLys [α-NH ₂ .TFA] [ε-CBz] was obtained as a pale yellow oil (46.04 g, 103% of theory because some methanol was still present).
¹ H nmr (300 MHz, CD ₃ OD) δ (ppm): 1.5-1.6 (m, 4H); 1.8-2.0 (m, 2H); 1.44 (s, 9H); 3.15 (t, J 6.8Hz, 2H) 3.71 (s, 3H); 3.88 (t, J 6.4Hz, 1H); 3.94 (d, J 17.6Hz, 1H); 4.08 (d, J 17.6Hz, 1H); 5.07 (s, 2H); 7.2- 7.4 (m, 5H).
LC / MS (hydrophilic / formate): ESI (+ ve) Found ^{[M + H] + m /} z = 352.1; ( as _{C 17 H 26 N 3 O 5} ) Calculated 352.2. Rf (minutes) = 12.3.

iii. MeOGlyLys [ε-CBz] [α -Lys] [Boc] 2 manufacturing
To a stirred solution of MeOGlyLys [α-NH ₂ .TFA] [ε-CBz] (46.0 g, 96.0 mmol) and triethylamine (24.3 g, 0.240 mol) in dimethylformamide (200 ml), DBL-OPNP (49.37 g, 0.106 mol) was added. After stirring for 17 hours at ambient temperature, a solution of glycine (3.98 g, 53.0 mmol) in water (50 ml) was added and the cloudy solution was stirred for 24 hours. Water (150 ml) was added to the well stirred mixture and a white solid began to precipitate. Next, flaky ice (200 g) was added and stirring was continued until the ice melted. The solid was collected by filtration, suspended in 5% aqueous sodium carbonate and sonicated for 0.5 hours. After semi-drying by suction, it was washed with 5% aqueous sodium carbonate solution (200 ml × 2) and then with water (200 ml × 3). The light yellow solid was then stirred in a new 200 ml 5% aqueous sodium carbonate solution and filtered to give a pale yellow powder. The solid was washed with water (200 ml × 2), suspended in fresh water (200 ml), and sonicated for 1.5 hours. After filtration and suction drying, 61.0 g of tan powder was obtained by drying under reduced pressure. Recrystallization from ethyl acetate gave MeOGlyLys [ε-CBz] [α-Lys] [Boc] ₂ (50.05 g, 77%) as a white solid.
¹ H nmr (300 MHz, CD ₃ OD) δ (ppm): 1.2-2.0 (m, 30H); 3.03 (t, J 6.6 Hz, 2H); 3.12 (t, J 6.8 Hz, 2H); 3.69 (s, 3H); 3.87 (d, J 17.6Hz, 1H); 4.00 (d, J 17.6Hz, 1H); 4.01 (dd, J 3.3, 7.4Hz, 1H); 4.38 (dd, J 5.4, 8.4Hz, 1H) 5.06 (s, 2H); 7.2-7.4 (m, 5H).
LC / MS (hydrophilic / formate): ESI (+ ve) measured value [M + H] ⁺ m / z = 680.0; calculated value (as C ₃₃ H ₅₄ N ₅ O ₁₀ ) 680.4; measured value [M + NH ₄ ] ⁺ m / z = 697.0; calculated (as C ₃₃ H ₅₇ N ₆ O ₁₀ ) 697.4. Rf (minutes) = 8.0.

iv. _{MeOGlyLys [ε-NH 2 .TFA]} [α-Lys] [Boc] 2 manufacturing
A solution of MeOGlyLys [ε-CBz] [α-Lys] [Boc] ₂ (680 mg, 1.00 mmol) and trifluoroacetic acid (77 μl, 1.0 mmol) in methanol (10 ml) under hydrogen at atmospheric pressure, 10% w / w Added to a suspension of palladium on charcoal (106 mg, 0.10 mmol Pd). The mixture was stirred at ambient temperature for 1 hour and then filtered through a bed of celite. Methanol was evaporated under reduced pressure, the residue redissolved in methanol (5 ml) and the solution passed through a 0.2 μm filter. Methanol was evaporated under reduced pressure to give MeOGlyLys [ε-NH ₂ .TFA] [α-Lys] [Boc] ₂ (640 mg, 97%) as a brittle white foam.
¹ H nmr (300 MHz, CD ₃ OD) δ (ppm): 1.2-2.0 (m, 30H); 2.93 (t, J 7.5Hz, 2H); 3.03 (t, J 6.6Hz, 2H); 3.72 (s, 3H); 3.89 (d, J 17.7Hz, 1H); 3.98 (dd, J 5.4, 9.0Hz, 1H); 4.02 (d, J 17.7Hz, 1H); 4.42 (dd, J 5.7, 8.4Hz, 1H) .
LC / MS (hydrophilic / formate): ESI (+ ve) Found ^{[M + H] + m /} z = 546.2; ( as _{C 25 H 48 N 5 O 8} ) Calculated 546.3. Rf (minutes) = 14.2.

v. Production of MeOGlyLys [Lys] 2 [Boc] 3 [ε, ε-CBz]
To a stirred solution of MeOGlyLys [ε-NH ₂ .TFA] [α-Lys] [Boc] ₂ (640 mg, 0.97 mmol) in dimethylformamide (10 ml), add PNPO-α-Boc-ε-CBz-Lys (535 mg 1.07 mmol). Triethylamine (340 μl, 2.43 mmol) was added and the solution was stirred at ambient temperature for 20 hours. A solution of glycine (40 mg, 0.54 mmol) in water (5 ml) was added and the cloudy solution was stirred for 2 hours. Dimethylformamide was evaporated under reduced pressure and the remaining oil was partitioned between ethyl acetate (20 ml) and a 3: 1 mixture of 5% aqueous sodium carbonate and saturated aqueous sodium chloride (20 ml). The aqueous phase is discarded, and the ethyl acetate phase is washed with a fresh sodium carbonate / sodium chloride mixture (20 ml × 3), then with 0.20 M hydrochloric acid (20 ml × 2) and then with a saturated aqueous sodium chloride solution (20 ml). Washed with. The ethyl acetate solution was dried (sodium sulfate), filtered and the solvent was evaporated under reduced pressure to give MeOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz] (818 mg, 93%) brittle white Obtained as a foam.
¹ H nmr (300 MHz, CD ₃ OD) δ (ppm): 1.2-2.0 (m, 45H); 3.03 (t, J 6.6 Hz, 2H); 3.12 (t, J 6.6 Hz, 2H); 3.19 (m, 3.71 (s, 3H); 3.88 (d, J 17.4Hz, 1H); 3.93-4.06 (m, 2H); 4.00 (d, J 17.7Hz, 1H); 4.38 (dd, J 5.4, 8.4Hz , 1H); 5.07 (s, 2H); 7.25-7.45 (m, 5H).
LC / MS (hydrophobic / formate): ESI (+ ve) measured [M + H] ⁺ m / z = 908.4; calculated (as C ₄₄ H ₇₄ N ₇ O ₁₃ ) 908.5; measured [M + NH ^{_{4] + m / z = 925.4}} ; ( as _{C 44 H 77 N 8 O 13} ) calculated 925.5. Rf (min) = 9.5.

vi. Production of HOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz]
MeOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz] (500 mg, 0.55 mmol) was dissolved in a solution of sodium hydroxide (44 mg, 1.10 mmol) in methanol (8 ml) and water (4 ml). The solution was stirred at ambient temperature for 4 hours and the solvent was evaporated under reduced pressure. The residue was dissolved in water (10 ml) and 1M potassium hydrogen sulfate (2 ml) was added. The resulting white precipitate was extracted into ethyl acetate (10 ml) and the aqueous phase was discarded. The ethyl acetate phase was washed with saturated aqueous sodium chloride solution (10 ml), dried (sodium sulfate), filtered, and the solvent was evaporated under reduced pressure to produce HOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz ] (481 mg, 96%) was obtained as an amorphous white solid.
¹ H nmr (300 MHz, CD ₃ OD) δ (ppm): 1.2-2.0 (m, 45H); 3.03 (t, J 6.6 Hz, 2H); 3.12 (t, J 6.6 Hz, 2H); 3.19 (m, 2H); 3.84 (d, J 18.0Hz, 1H); 3.95-4.07 (m, 2H); 3.97 (d, J 17.7Hz, 1H); 4.39 (dd, J 5.4, 8.4Hz, 1H); 5.07 (s , 2H); 7.25-7.38 (m, 5H).
LC / MS (hydrophobicity / TFA): ESI (+ ve ) Found ^{[M + H] + m /} z = 894.3; ( as _{C 43 H 72 N 7 O 13} ) Calculated 894.5. Rf (min) = 8.4.
(Example 37)

Preparation of BHALys [GlyLys] ₂ [Lys] ₄ [Boc] ₆ [ε, ε-CBz] ₂ (C ₁₀₅ H ₁₆₃ N ₁₇ O ₂₅ MW2063.5) The synthesis is illustrated in FIG. HOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz] (536 mg, 0.60 mmol), BHALys [NH ₂ .TFA] ₂ (135 mg, 0.250 mmol), 4,4-dimethylaminopyridine (7.3 mg, 60 μmol) ) And triethylamine (210 μl, 1.50 mmol) in dimethylformamide (10 ml) was added EDCI (0.90 mmol). The solution was stirred at ambient temperature for 15 hours and then the volatile components were removed under reduced pressure. Silica gel chromatography (methanol / dichloromethane gradient) gave BHALys [GlyLys] ₂ [Lys] ₄ [Boc] ₆ [ε, ε-CBz] ₂ (245 mg, 47%). A small sample (20 mg) was treated with acetic acid / TFA in a conventional manner to obtain analytical data.
LC / MS (philic / TFA): ESI (+ ve) found [M + H] ⁺ m / z = 1463.2; calculated (as C ₇₅ H ₁₁₆ N ₁₇ O ₁₃ ) 1462.9.
(Example 38)

_{BHALys [Lys] 16 [α-} PEG 570] 16 [COCH 3] 16 production of
The _{BHALys [Lys] 16 [α-} PEG 570] 16 [NH 2 .TFA] 16 in DMF and TEA (NH ₂ per 3 eq) which was stirred and added acetic anhydride (NH ₂ per 1.5 eq) The reaction was stirred overnight. The reaction was concentrated and the residue was purified by size exclusion chromatography on Sephadex G-25 with water as the eluent to give BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [COCH ₃ ] ₁₆ .
(Example 39)

Dendrimer lymphatic targeting rats were cannulated into the thoracic duct (using the method described in M Boyd et al. (2003) Journal of Pharmacology and Toxicology Methods 49: 115-120) and the right jugular vein (for saline infusion). Rats were allowed to recover overnight and were constantly fed food and water. Dendrimers (Lys ₁₆ (PEG ₂₀₀ ) ₃₂ , Lys ₁₆ (PEG ₅₇₀ ) ₃₂ , Lys ₁₆ (PEG ₂₀₀₀ ) ₃₂ or Lys ₁₆ (BS) ₃₂ ) in 5 mg / kg dose (10 mg / ml, 50 μl per 100 g body weight) Was administered subcutaneously by injection onto the starboard. Lys ₁₆ (BS) ₃₂ was the non-PEGylated control dendrimer. Lymph drainage into the thoracic duct was collected over 30-48 hours and scintillation counting was performed for tritium radiolabel.

The results of this study demonstrated that up to 40% of the subcutaneous dose of PEGylated poly-L-lysine dendrimer can be taken up into the lymph within 48 hours of injection administration (Figure 23). This depends on the size of the dendrimer, 38.5 ± 0.7% (mean ± SD, n = 3) of Lys ₁₆ (PEG ₂₀₀₀ ) ₃₂ (68 kDa) is taken up by the lymph in 48 hours and 28.8 ± 6.6% in 30 hours While Lys ₁₆ (PEG ₅₇₀ ) ₃₂ (22 kDa) was recovered in the lymph, only 3.8% (n = 1) of Lys ₁₆ (PEG ₂₀₀ ) ₃₂ (11 kDa) was recovered in the thoracic lymph. Non-PEGylated benzenesulfonate dendrimer (11 kDa) was only 1.7 ± 1.5% uptake into the lymph in 30 hours (mean ± SD, n = 3), so PEGylation helped to increase the uptake of dendrimer into the lymph It was. At the time of sacrifice, a total of approximately 0.3% of the dose of Lys ₁₆ (PEG ₅₇₀ ) ₃₂ and Lys ₁₆ (PEG ₂₀₀₀ ) ₃₂ dendrimer was recovered in the right popliteal and iliac lymph nodes. This represents that 2-3% dose / g was recovered in the lymph nodes and the concentration of radiolabel typically recovered in major organs 30-168 hours after IV administration (1.5% dose / g tissue Considerably higher than).

In summary, large (> 20 kDa) PEGylated dendrimers were taken up by lymphatics in the case of small (11 kDa) PEGylated or non-PEGylated dendrimers, whereas significant amounts were taken up into regional lymph vessels after subcutaneous administration. The amount was much less. In the case of non-PEGylated substances, this may also reflect a decrease in drainage from the injection site. Only a small percentage of the dose was collected in the regional lymph nodes 30 to 48 hours after subcutaneous administration, but given the small mass of the lymph nodes (approximately 0.1 to 0.2 g), Represents a relatively high dendrimer concentration in the lymph nodes. PEGylation of PLL dendrimers with larger PEG groups can further increase lymph recovery. These results highlight the potential of PEGylation to increase drug-dendrimer complex uptake into the lymph following subcutaneous administration.
(Example 40)

Pharmacokinetics of 50% PEG ₅₇₀ capped poly-L-lysine dendrimer The following studies show that 1) partial surface PEGylation and 2) complete capping of the dendrimer surface with a combination of PEG and drug / acetyl groups is This was done to determine how it affects dendrimer pharmacokinetics after kg IV administration.

The following tritiated (G3) dendrimers were used in this study:
Lys ₁₆ (NH ₂ ) ₃₂ (MW 4.1 kDa), BHALys [ ³ H-Lys] ₁₆ [NH ₂ ] ₃₂ , also referred to as uncapped dendrimer in the text;
Lys ₁₆ (PEG ₅₇₀ ) ₃₂ (MW 23 kDa), BHALys [ ³ H-Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ , also referred to herein as a fully PEGylated dendrimer;
Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (NH ₂ ) ₁₆ (MW 13.3 kDa), BHALys [ ³ H-Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-NH ₂ ] ₁₆ , also referred to herein as a semi-PEGylated dendrimer;
Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (CH ₃ ) ₁₆ (MW 14 kDa), BHALys [ ³ H-Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-COCH ₃ ] ₁₆ , also referred to herein as a semi-acetylated dendrimer;
Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (MTX amide) ₁₆ ^* (MW 22.5 kDa), BHALys [ ³ H-Lys] ₈ [Su (NPN) ₂ ] ₁₆ [PEG ₅₇₀ ] ₁₆ [α-tBu-MTX] ₁₆ , in text Also referred to as MTX dendrimer, in which case MTX is methotrexate.
^* Methotrexate was conjugated to the non-PEGylated site via a stable amide linker.

Method
SD rat (about 300g) with 5mg / kg dose of tritiated dendrimer (in 1ml saline)
Administration was by direct intravenous infusion for 2 minutes through an indwelling cannula in the right jugular vein. A t0 blood sample was then collected from the indwelling cannula of the right carotid artery (0.2 ml) to assess Cp0 (the concentration of dendrimer in plasma at the end of intravenous infusion). Thereafter, blood samples were collected in heparinized tubes at 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, 480, 1440 and 1800 minutes. After centrifugation of the blood sample, 100 μl of plasma was mixed with 1-2 ml of Starscint in a 6 ml scintillation tube and counted for tritium radioactivity. Urine was collected at time intervals of 0-8 hours, 8-24 hours, and 24-30 hours after dendrimer administration. A portion of urine (100 μl each) was similarly counted for tritium radioactivity. Urine and plasma samples were analyzed by size exclusion chromatography on a Superdex 75 SEC column eluting with 50 mM PBS + 0.3 M NaCl (pH 3.5). Fractions eluting from the column were collected at 1 minute (0.5 ml) intervals, mixed with 2 ml Starscint in a 6 ml scintillation tube and scintillation counted for tritium radioactivity.

  The potential for binding to blood vessels or tissue surfaces was estimated for each dendrimer by measuring binding to liver homogenate as a surrogate as follows.

To remove most of the blood in the blood vessels, the liver from anesthetized rats was perfused with saline. The liver was then isolated and ground with a glass homogenizer in 1: 1 w / v saline. Rats were sacrificed by lethal injection of Lethabarb by cardiac puncture. When the liver was visibly homogenized, approximately 1 ml of liver homogenate was placed in a microfuge tube and centrifuged at 3500 rpm for 5 minutes. The supernatant was removed and an additional 500 μl of saline was added to each tube. Each tube was briefly vortexed and centrifuged again. This step ensures that as much soluble protein as possible is removed from the homogenate to minimize the amount of protein that can bind to the dendrimer and count as unbound dendrimer. . Add 500 μl of saline to each tube and add 50 μg of Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (CH ₃ ) ₁₆ , Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (NH ₂ ) ₁₆ or Lys ₈ (D-lys) ₁₆ to each tube. Added to. D-lys dendrimer was used as a cationic uncapped dendrimer control because it does not undergo rapid metabolism that can affect the results of binding experiments. Each tube was incubated on a rotary mixer at 37 ° C. for 30 minutes, after which the tube was centrifuged again. The supernatant was collected and analyzed for “unbound” dendrimers. The remaining liver homogenate was solubilized as described elsewhere to ensure that the remaining dendrimer bound to liver tissue.

Result :
Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (NH ₂ ) ₁₆
After administration of the half-PEG dendrimer, plasma radiolabel decreased rapidly with a half-life of 8.6 ± 0.3 minutes over the first hour (FIG. 24B). For comparison, the plasma profile of fully uncapped (Lys ₁₆ (NH ₂ ) ₃₂ ) dendrimer is shown in FIG. 24A. The reason for the difference in initial plasma / distribution kinetics between fully uncapped and semi-PEGylated dendrimers will be apparent from tissue binding data (Table 13). That is, uncapped dendrimers are thought to bind rapidly to tissues or vasculature (tested using liver homogenate as a surrogate) and as a result are almost immediately removed from plasma. In contrast, even partially PEGylated material has much lower tissue or vascular binding activity.

  After 1 hour, the decrease in plasma radiolabel dramatically slowed down and the terminal half-life (calculated over 6-30 hours) was 22.1 ± 2.1 hours. Size exclusion chromatography (FIG. 25A) suggests the presence of tritiated lysine in plasma up to 2 hours after administration, and also shows that the major molecular species present in plasma are significantly larger than dendrimers. Thus, a terminal half-life of about 1 day probably reflects the clearance of plasma proteins produced by reuptake of free tritiated lysine.

Plasma pharmacokinetics and SEC profiles for fully uncapped species suggest that after 30 minutes, a constant “feed” of lysine can be used to drive plasma protein synthesis (ie 6-30 post-dose). Time does not reveal a clear decrease in plasma radiolabel, plasma profiles over time are not listed here). However, plasma pharmacokinetics and SEC profiles obtained from semi-PEGylated dendrimers suggest that although a similar degradation and reuptake process appears to have occurred, it appears to have occurred in a shorter period of time. This is because reuptake products are eliminated with a half-life reflecting the turnover rate of albumin (approximately 24 hours). This relatively short period of supply of tritiated lysine to drive protein biosynthesis is consistent with the relatively easy renal clearance observed over the 30 hour sampling period, and half PEG 73.5 ± 2.8% of the administered tritium associated with dendrimer was recovered in the urine. This is in stark contrast to the recovery of injected radiolabels from Lys ₁₆ (NH ₂ ) ₃₂ and Lys ₁₆ (PEG ₅₇₀ ) ₃₂ (approximately 5% and 40%, respectively). Molecular species identified in urine co-eluted with complete dendrimers in SEC (Figure 25B).

Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (CH ₃ ) ₁₆
The initial plasma profile of the radiolabel derived from the half-acetylated dendrimer was similar to that of the half-PEGylated dendrimer (half-life = 9.3 ± 0.42 min) (FIG. 24). However, unlike half-PEGylated dendrimers, the terminal half-life of the half-acetylated dendrimer was shorter and closer to the terminal half-life of the fully PEGylated molecular species (at 13.9 ± 0.3 hours for the half-acetylated dendrimer). In contrast, the complete PEGylated dendrimer is 9.45 ± 0.42 hours). This is probably due to the slower rate of core metabolism for half-acetylated dendrimers than for half-PEGylated dendrimers (FIG. 27A). The plasma SEC profile shows that at t0, all of the plasma radiolabel is believed to be due to the complete dendrimer. Plasma collected 2 hours after dosing showed a small peak attributed to the complete dendrimer and a broad peak 20 minutes (this peak eluted 2 minutes before the complete dendrimer), but free lysine was I didn't see it. In the case of semi-PEGylated dendrimers, most of the injected radiolabel was excreted in the urine for 30 hours (72.3 ± 1.2%) and most of it was excreted unchanged. Several small molecular weight species believed to be due to products of dendrimer metabolism were identified in the 8-24 hour urine (FIG. 27B, note that the scale is different for 8-24 hours).

Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (MTX amide) ₁₆
Although the molecular weight of the MTX dendrimer was similar to that of the fully PEGylated dendrimer, its plasma profile was more similar to that of the semi-acetylated dendrimer. The initial plasma half-life was 15.4 ± 2.4 minutes, which was slightly slower than the other half-capped dendrimer. The terminal elimination half-life is essentially the same as the fully PEGylated dendrimer (9 ± 0.2 hours), probably reflecting that terminal phase plasma clearance depends on the total molecular weight.

  Less than one third of the tritiated dendrimer dose was eliminated via urine during the 30 hour period (29 ± 3.4). Most of them were excreted in the first 8 hours after IV administration. The amount of MTX dendrimer excreted in urine was much lower (42.9 + 2.7%) than 1) the other half-capped dendrimer and 2) the fully PEGylated dendrimer. Although we cannot fully explain this small amount of renal exclusion, 1) the large size of the dendrimer hindered renal exclusion to some extent, 2) the remainder of the dose concentrated in the reticuloendothelial (RES) organs And 3) some of the dendrimers may have been retained in the organ by interaction with the folate binding site (because MTX is a competitive antagonist of folate at the folate receptor) .

Conclusion :
1) Lysine dendrimers with 50% surface capping by PEG ₅₇₀ (but 50% of surface sites remain uncapped) are cleared from plasma relatively quickly and undergo free degradation to release free lysine It seems to do. However, they do not appear to exhibit as much vascular binding as the fully uncapped species.
2) Acetylation of uncapped sites on dendrimers with 50% surface PEGylation reduces dendrimer biodegradation compared to dendrimers where 50% of surface amines remain uncapped, but plasma The initial clearance / distribution speed from is basically the same.
3) Lysine dendrimers with 50% PEG capping groups and 50% MTX capping groups on the surface show an initial plasma profile similar to semi-acetylated dendrimers.
4) Lysine dendrimers with 50% PEG capping groups and 50% MTX capping groups on the surface are recovered in urine after IV administration compared to similar sized fully PEGylated dendrimers or 50% acetylated systems The dose ratio is small. This probably reflects the increased uptake by RES caused by the interaction of MTX with phagocytic cells or folate receptors in RES.

References

1． Frechet, JMJ “Dendrimers and other dendritic macromoleucles: From building blocks to functional assemblies in nanoscience and nanotechnology” J. Polym. Sci. A. 2003, 41, 3713-3725.
2． Beezer, AE; King, ASH; Martin, IK; Mitchel, JC; Twyman, LJ; Wain, CF “Dendrimers as potential drug carriers; encapsulation of acidic hydrophobes within water soluble PAMAM derivatives” Tetrahedron 2003, 59, 3873-3880.
3． Kojima, C; Kono, K .; Maruyama, K .; Takagishi, T. "Synthesis of polyamidoamine dendrimers having poly (ethylene glycol) grafts and their ability to encapsulate anticancer drugs" Bioconjug. Chem. 2000, 11, 910-917.
Four. Tajarobi, F .; El-Sayed, M .; Rege, BD; Polli, JE; Ghandehari, H. “Transport of poly amidoamine dendrimers across Madin-Darby canine kidney cells” Int. J. Pharm. 2001, 215, 263- 267.
Five. El-Sayed, M .; Ginski, M .; Rhodes, C .; H., G. “Transepithelial transport of poly (amidoamine) dendrimers across Caco-2 cell monolayers.” J. Control. Release 2002, 81, 355- 365.
6． Malik, N .; Wiwattanapatapee, R .; Klopsch, R .; Lorenz, K .; Frey, H .; Weener, JW; Meijer, EW; Paulus, W .; Duncan, R. “Dendrimers: Relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of I-125-labelled polyamidoamine dendrimers in vivo '' J. Control. Release 2000, 65, 133-148.
7． Wiwattanapatapee, R .; Carreno-Gomez, B .; Malik, N .; Duncan, R. “Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: A potential oral delivery system?” Pharm. Res. 2000, 17, 991- 998.
8． Jevprasesphant, R .; Penny, J .; Jalal, R .; Attwood, D .; McKeown, NB; D'Emanuele, A. “The influence of surface modification on the cytotoxicity of PAMAM dendrimers” Int. J. Pharm. 2003 , 252, 263-266.
9． Sakthivel, T .; Toth, I .; Florence, AT. “Distribution of a lipidic 2.5 nm diameter dendrimer carrier after oral administration” Int. J. Pharm. 1999, 183, 51-55.
Ten. Florence, AT .; Sakthivel, T .; Toth, I. "Oral uptake and translocation of a polylysine dendrimer with a lipid surface" J. Control. Release 2000, 65, 253-259.
11． Jevprasesphant, R .; Penny, J .; Attwood, D .; D'Emanuele, A. “Transport of dendrimer nanocarriers through epithelial cells via the transcellular route” J. Control. Release 2004, 97, 259-267.
12． Gillies, ER; Dy, E .; Frechet, JMJ; Szoka, FC, Jr. “Biological Evaluation of Polyester Dendrimer: Poly (ethylene oxide)“ Bow-Tie ”Hybrids with Tunable Molecular Weight and Architecture” Mol. Pharm. 2005, 2, 129-138.
13. Kobayashi, H .; Kawamoto, S .; Saga, T .; Sato, N .; Hiroga, A .; Konishi, J .; Togashi, K .; Brechbiel, MW “Micro-MR angiography of normal and intratumoral vessels in mice using dedicated intravascular MR contrast agents with high generation of polyamidoamine dendrimer core: Reference to pharmacokinetic properties of dendrimer-based MR contrast agents '' J. Magn. Reson. Imaging 2001, 14, 705-713.
14. Kobayashi, H .; Wu, CC; Kim, MK; Paik, CH; Carrasquillo, JA; Brechbiel, MW "Evaluation of the in vivo biodistribution of indium-111 and yttrium-88 labeled dendrimer-1 B4M-DTPA and its conjugation with anti-Tac monoclonal antibody '' Bioconjug. Chem. 1999, 10, 103-111.
15． Kobayashi, H .; Saga, T .; Kawamoto, S .; Sato, N .; Hiroga, A .; Ishimori, T .; Konishi, J .; Togashi, K .; Brechbiel, MW “Dynamic micro-magnetic resonance imaging of liver micrometastasis in mice with a novel liver macromolecular magnetic resonance contrast agent DAB-Am64- (1B4M-Gd) (64) '' Cancer Res. 2001, 61, 4966-4970.
16． Kobayashi, H .; Kawamoto, S .; Saga, T .; Sato, N .; Hiroga, A .; Ishimori, T .; Konishi, J .; Togashi, K .; Brechbiel, MW “Positive effects of polyethylene glycol conjugation to generation-4 polyamidoamine dendrimers as macromolecular MR contrast agents '' Magn. Reson. Med. 2001, 46, 781-788.
17． Marionum, LD; Campion, BK; Koo, M .; Shargill, N .; Lai, JJ; Marumoto, A .; Sontum, PC “Gadolinium (III) DO3A macrocycles and polyethylene glycol coupled to dendrimers-Effect of molecular weight on physical and biological properties of macromolecular magnetic resonance imaging contrast agents '' J. Alloys Compounds 1997, 249, 185-190.
18． McCarthy, TD; Karellas, P .; Henderson, SA; Giannis, M .; O'Keefe, DF; Heery, G .; Paul, JRA; Matthews, BR; Holan, G. “Dendrimers as Drugs: Discovery and Preclinical and Clinical Development of Dendrimer-Based Microbicides for HIV and STI Prevention '' Mol. Pharm. 2005, 2, 312-318.

  It will be understood that the invention disclosed and defined herein extends to each of the individual features referred to in the text or drawings or two or more alternative combinations of individual features apparent from the text or drawings. Any of these different combinations constitute various alternative aspects of the invention.

FIG. 1A—Chemical structure of Lys ₈ (NH ₂ ) ₁₆ dendrimer BHALys [Lys] ₈ [NH ₂ ] ₁₆ . Each of the eight L-Lys _8- terminal lysine groups imparts two positive charges at physiological pH. Since the D-Lys group attached to the Lys ₈ core was not radiolabeled, the site of ³ H radiolabeling was on the Lys ₈ layer of Lys ₁₆ dendrimer. FIG. 1B—Chemical structure of L-Lys ₁₆ (NH ₂ ) ₃₂ dendrimer BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ . Each of the 16 L-Lys _16- terminal lysine groups imparts two positive charges at physiological pH. An asterisk on the terminal lysine and in the inset highlights the position of the ³ H radiolabel on the surface lysine group. R represents the attachment of surface lysine to the dendrimer core. Plasma concentration of cationic ³ H-dendrimer (mean ± SD, n = 3) after intravenous administration to rats at a dose of 5 mg / kg. The closed symbol represents administration of BHALys [Lys] ₈ [NH ₂ ] ₁₆ and the hollow symbol represents BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ . Data are expressed as ng equivalent / ml of dendrimer administered. In a pilot study, plasma concentration of cationic ³ H-dendrimer (n = 1) after intravenous administration to rats at higher doses. Closed symbols indicate data obtained after administration of 24.3 mg / kg BHALys [Lys] ₈ [NH ₂ ] ₁₆ ; hollow symbols represent 22.3 mg / kg BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ Data obtained after administration is represented. Data are expressed as ng equivalent / ml of dendrimer administered. Whole blood concentration of cationic ³ H-dendrimer after intravenous administration to rats at a dose of 5 mg / kg (mean ± SD, n = 3). The closed symbol represents administration of BHALys [Lys] ₈ [NH ₂ ] ₁₆ and the hollow symbol represents BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ . Data are expressed as ng equivalent / ml of dendrimer administered. Plasma concentrations of BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ , BHALys [Lys] ₈ [D-Lys] ₁₆ [NH ₂ ] ₃₂ and L-lysine after intravenous administration of 5 mg / kg to rats (mean ± SD, n = 3). The closed circle represents the administration of BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ , the hollow circle represents BHALys [Lys] ₈ [D-Lys] ₁₆ [NH ₂ ] ₃₂ , and the closed triangle represents L-lysine. To express. Data are expressed as ng equivalent / ml of dendrimer or lysine administered. Size exclusion chromatography (SEC) profile obtained using a Superdex Peptide 10/300 GL size exclusion column. Panel A-BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ and lysine after 1 hour incubation at room temperature in blank plasma with heparin. The elution volume and elution profile were identical to those obtained for BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ and lysine (profile not shown). Panel B- Plasma sample taken immediately after intravenous administration of BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ (t = 0). Panel _{C-BHALys [Lys] 16 [} NH 2] After ₃₂ 3 hours intravenous administration (open symbols) and after 6 hours (closed symbols) to collected plasma samples. Plasma sample collected 30 hours after intravenous administration of panel DL-lysine. Elution profile of plasma radiolabel from Superdex 75 HR 10/30 size exclusion column (as elution DMP). Closed symbols represent elution profiles for plasma samples collected 6 hours after intravenous administration of BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ (scale on left Y axis). The hollow symbol represents the elution profile for the plasma sample collected 30 hours after intravenous infusion of L-lysine (scale on the right Y axis). The elution times are shown at the top of the graph for protein standards of various molecular weights, BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ and lysine. The void volume of the column was determined by injecting Blue Dextran 2000 (molecular weight 2000 kDa) and is shown at the bottom of the figure (V ₀ ). Distribution of residual ³ H in tumor organs 30 hours after intravenous administration of cationic ³ H-dendrimer to rats at 5 mg / kg. Panel A is data expressed as% of injected radiolabel, and panel B represents data as% of injected radiolabel per gram of tissue (mean ± SD, n = 3). The closed symbol represents the tissue biodistribution for BHALys [Lys] ₈ [NH ₂ ] ₁₆ and the hollow symbol represents the tissue biodistribution for BHALys [Lys] ₁₆ [NH ₂ ] ₃₂ . The shaded (gray) symbol represents the tissue biodistribution for BHALys [Lys] ₈ [D-Lys] ₁₆ [NH ₂ ] ₃₂ . Schematic representation of selected topological isomers of a lysine dendritic motif according to a preferred embodiment of the present invention, having end groups A and B in a 1: 1 surface ratio and having 5 generational building units from apical F. A and B represent two different protecting groups or organic groups, and F represents an incomplete carboxylate at the apex. BHALys [Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ (closed circle), BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na) after administration of 5 mg / kg IV to rats ] Plasma concentration-time profiles of ₃₂ (hollow circle) and BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ (triangle). BHALys [Lys] ₈ [CO-4-Ph (SO ₃ Na)] ₁₆ (black), BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ (light gray), BHALys [Lys ] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ (dark gray) or BHALys [Lys] ₁₆ [CO-CH ₂ CH ₂ (CO ₂ Na)] ₃₂ (white) IV administered 30 Biodistribution of injected ³ H after time. Panel A-% of injected ³ H present for each organ. Panel B-% of ³ H injection present per gram of tissue. On a Superdex 75 column of BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ and BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ in plasma and urine Size exclusion profile. Panel A—SEC profile of BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ incubated for 1 hour in PBS (closed circle) or in fresh plasma (hollow circle). Panel B—SEC profile of BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ incubated for 1 hour in PBS (closed circle) or in fresh plasma (hollow circle). SEC profiles of BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ in plasma at panels C-t0 (closed circle) and 2 hours (hollow circle). SEC profile of BHALys [Lys] ₁₆ [CO-3,5-Ph (SO ₃ Na) ₂ ] ₃₂ in plasma at panel D-t0 (closed circle) or 2 hours (hollow circle). Panel E-0 SEC profiles of BHALys [Lys] ₁₆ [CO-4-Ph (SO ₃ Na)] ₃₂ in urine (closed circles) and 0-24 hour urine (hollow circles). BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ (closed circle), BHALys [Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ (hollow circle), BHALys [Lys] ₈ [PEG ₂₀₀₀ ] ₁₆ (closed triangle) and BHALys [ Lys] ₁₆ [PEG ₂₀₀₀ ] ₃₂ (hollow triangle) plasma concentration-time profile. Data on BHALys [Lys] ₈ [PEG ₂₀₀ ] ₁₆ are not shown. This is because the disappearance is extremely rapid and is hidden in the data relating to BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ . BHALys [Lys] ₁₆ [PEG ₂₀₀₀ ] ₃₂ (black bar, 7 days), BHALys [Lys] ₈ [PEG ₂₀₀₀ ] ₁₆ (gray bar, 5 days) and BHALys [Lys] ₁₆ [PEG ₅₇₀ ] after IV administration _{Biodistribution of 32} (white bar, 30 hours). The upper panel represents the% of the injected dose of ³ H recovered in each organ, lower panels represent the% of the injected dose of ³ H recovered per each tissue 1 g. Only 0.1% of the ₃₂ doses of BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ were recovered in the kidney 30 hours after IV administration, whereas in any rat receiving BHALys [Lys] ₈ [PEG ₂₀₀ ] ₁₆ ³ H was not detected (data not shown). ³ H-labeled BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ in plasma after administration of 5 mg / kg IV (Panel A; t0, hollow square, t = 1 hour, closed circle, closed circle, t = 4 hours , Hollow circle), BHALys [Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ (Panel B; 24 hours), BHALys [Lys] ₈ [PEG ₂₀₀₀ ] ₁₆ (Panel C; 48 hours) and BHALys [Lys] ₁₆ [PEG ₂₀₀₀ ] Size exclusion profile on Superdex 75 column for ₃₂ (Panel D; 48 hours). Arrows indicate complete dendrimer retention time. BHALys [Lys] ₁₆ [PEG ₂₀₀ ] ₃₂ (Panel A; 0-4 hr urine, closed circle, 8-24 hr urine, hollow circle) and BHALys [Lys] ₁₆ [PEG ₅₇₀ ] ₃₂ (Panel B; 8 Size exclusion profile of ³ H excreted in urine after IV administration (~ 24 hours urine). Arrows indicate complete dendrimer retention time. Reaction scheme 3 for producing BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-MTX] ₁₆ . Reaction scheme 5 for producing BHALys [Lys] ₁₆ [α-PEG ₅₇₀ ] ₁₆ [ε-COCH ₂ CH ₂ CO-taxol]. FIG. 19A-BHALys [Lys] ₁₆ [ε, ε-PEG ₅₇₀ ] ₈ [COCH ₂ CH ₂ CO-Taxol] Reaction Scheme 6 for preparing ₂₄ (Part 1). FIG. 19B—Reaction Scheme 6 (Part 2) for producing BHALys [Lys] ₁₆ [ε, ε-PEG ₅₇₀ ] ₈ [COCH ₂ CH ₂ CO-Taxol] ₂₄ . Reaction scheme 7 for producing PEG ₁₇₁₆ -CO ₂ H, PEG ₂₆₄₅ -CO ₂ H, PEG ₃₉₇₄ -CO ₂ H. Illustration of the synthesis of Example 36 including the following steps: i. Reaction of MeOGly [NH ₂ .HCl] with PNPO-Lys-α-Boc-ε-CBz and triethylamine in DMF; ii. Reaction of MeOGlyLys [α-Boc] [ε-CBz] with 1: 1 TFA / AcOH; iii. Reaction of MeOGlyLys [α-NH ₂ .TFA] [ε-CBz] with DBL-OPNP and triethylamine in DMF; iv. Reaction of MeOGlyLys [α-Lys] [Boc] ₂ [ε-CBz] with catalytic amounts of palladium on charcoal and 1 equivalent of TFA in methanol; v. Reaction of MeOGlyLys [α-Lys] [Boc] ₂ [ε-NH ₂ .TFA] with PNPO-Lys-α-Boc-ε-CBz and triethylamine in DMF; vi. Reaction of MeOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz] with sodium hydroxide in MeOH / H ₂ O, followed by reaction with aqueous potassium hydrogen sulfate solution. Illustration of the synthesis of Example 37 including the following steps: i. Reaction of BHALys [NH ₂ .TFA] ₂ with HOGlyLys [Lys] ₂ [Boc] ₃ [ε, ε-CBz], excess EDCI and HOBt in DMF; ii. Reaction of BHALysBHALys [GlyLys] ₂ [Lys] ₄ [Boc] ₆ [ε, ε-CBz] ₂ with 1: 1 TFA / AcOH. Cumulative recovery over time in thoracic lymph fluid at subcutaneous doses of PEGylated dendrimers (black symbols) or non-PEGylated benzenesulfonate dendrimers (white symbols). Results are mean ± sd (n = 1-3). Plasma concentration-time profiles of PEGylated and uncapped Lys ₁₆ dendrimers. Panel A shows the initial reduction in plasma concentrations of fully PEGylated, uncapped and semi-PEGylated dendrimers. Data are expressed as mean ± sd (N = 2-3). SEC profiles of plasma (A) and urine (B) collected from rats dosed with 5 mg / kg tritiated (G3 layer) Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (NH ₂ ) ₁₆ . The complete dendrimer elution time is indicated by an arrow. The product eluting at 18 and 21 minutes is the lysine reuptake product. The peak eluting at 43 minutes is labeled lysine. SEC profile of plasma collected from rats dosed with Lys ₁₆ (NH ₂ ) ₃₂ (6 hour sample) and L-lysine (30 hour sample). The 17 and 20 minute peaks are lysine reuptake products. The peak at 41 minutes is tritiated lysine. SEC profiles of plasma and urine collected from rats dosed with 5 mg / kg tritiated (G3 layer) Lys ₁₆ (PEG ₅₇₀ ) ₁₆ (CH ₃ ) ₁₆ . The complete dendrimer elution time is indicated by an arrow. The product eluting at 18 minutes is a lysine reuptake product. The peak eluting at 20 minutes is probably an anomalous peak associated with the complete dendrimer found in all capped molecular species. The peak eluting at 43 minutes is labeled lysine. The peak eluting at 30-40 minutes is probably the core degradation product.

Claims (49)

A polymer with controlled end group stoichiometry, comprising a surface layer, at least one sub-surface layer, and a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and A second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule,
Macromolecules containing at least two end groups containing (wherein end group stoichiometry refers to the number and type of end groups).   2. The polymer according to claim 1, further indicating a controlled topology, wherein the topology is one end group and another end group in terms of its connection to the surface layer or subsurface layer of the polymer. Represents the relationship).   2. The macromolecule of claim 1, wherein the second end group is selected from one or more of the following: a pharmaceutically active agent and / or a moiety that modifies the plasma half-life of the macromolecule; one or more cell types or tissues A moiety that facilitates targeting of a pharmaceutically active agent and / or macromolecule to a type; and a moiety that facilitates incorporation of a pharmaceutically active agent and / or macromolecule into one or more cell types or tissue types.   4. The polymer of claim 3, wherein the second end group is selected to increase the plasma half-life of the pharmaceutically active agent.   4. The polymer according to claim 3, wherein the second terminal group is a ligand for a cell surface receptor.   4. The polymer according to claim 3, wherein the second terminal group is polyethylene glycol (PEG) or polyethyloxazoline.   4. The polymer according to claim 3, wherein the second terminal group is a residue of folic acid or a folic acid derivative.   2. The polymer of claim 1, further comprising one or more linker moieties, wherein the first and / or second end groups are attached to the polymer framework via the linker moieties.   9. The polymer according to claim 8, wherein the linker moiety is cleavable.   9. The polymer of claim 8, wherein the linker moiety is selected from the group consisting of an amide linker, hydrazone, oxime and imine linker, ester linker, peptide linker, glutaraldehyde linker, PEG-peptide linker, disulfide linker and thymidine linker. .   The second end group is a moiety that facilitates targeting of the pharmaceutically active agent and / or macromolecule to one or more cell types or tissue types and the pharmaceutically active agent and / or to one or more cell types or tissue types. 2. The polymer of claim 1, wherein the polymer is selected from moieties that facilitate uptake of the polymer, and wherein the one or more linker moieties comprise PEG.   Pharmaceutically active agent is an acetoneemia preparation; anesthetic, antacid; antibody; antifungal; antiinfective; antimetabolite; antimitotic; antiprotozoal; antiviral drug; Dilators and expectorants; Cardiovascular drugs; Contrast agents; Diuretics; Growth hormones; Hematopoietics; Hormone replacement therapies; Immunosuppressants; Hormones and hormone analogs; Minerals; Dietary supplements and nutrients; Ophthalmic drugs; Drugs; Respiratory drugs; Transplant-related products; Vaccines and adjuvants; Anabolic agents; Analgesics; Anti-arthritic drugs; Anticonvulsants; Antihistamines; Anti-inflammatory drugs; Antimicrobial drugs; Antiparasitic drugs; Drugs; Blood and blood substitutes; Cancer drugs and cancer-related drugs; Central nervous system drugs; Contraceptives; Diabetes drugs; Infertility drugs; Growth-promoting drugs; Hemostatic drugs; Immunostimulants; Muscle relaxants; Natural products An anti-obesity drug; Osteoporotic disease agents; peptides and polypeptides; sedatives and tranquilizers; urine acidifying drugs; is selected from the group consisting of and vitamins, polymers according to claim 1.   13. The polymer of claim 12, wherein the pharmaceutically active agent is selected from the group consisting of an antimetabolite; an anti-mitotic agent; an anti-inflammatory agent; an anti-obesity agent; and an immunosuppressive agent.   14. The polymer of claim 13, wherein the pharmaceutically active agent is selected from the group consisting of methotrexate; taxol; indomethacin; xenical; and cyclosporine. A macromolecule with controlled end group stoichiometry, comprising a surface layer, at least one subsurface layer, and a first end group (residue of a peptide or protein, derivative or precursor thereof) One terminal group is attached to a selected single attachment point on the polymer), and at least two containing a second terminal group selected to modify the pharmacokinetics of the peptide or protein and / or polymer Macromolecules containing one end group (in this case, end group stoichiometry refers to the number and type of end groups). A polymer comprising controlled end group stoichiometry and comprising at least one lysine or lysine analog dendritic motif having a surface layer and at least one sub-surface layer,
A first terminal group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and at least two comprising a second terminal group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule Macromolecules containing one end group (in this case, end group stoichiometry refers to the number and type of end groups).   17. The polymer of claim 16, wherein the lysine dendrimer polymer further exhibits a controlled topology (where the topology is a terminal group in terms of its connection to the surface layer or sub-surface layer of the polymer). And the relationship to another terminal group).   17. The polymer according to claim 16, wherein the second terminal group is polyethylene glycol (PEG) or polyethyloxazoline.   17. The polymer according to claim 16, wherein the second terminal group is a residue of folic acid or a folic acid derivative.   17. The polymer of claim 16, wherein the pharmaceutically active agent is selected from the group consisting of methotrexate; taxol; indomethacin; xenical; and cyclosporine. With controlled capping group stoichiometry, the formula:
Core [repeating unit] _m [surface building unit] _n [capping group 1] _p [capping group 2] _q
[Where:
The core is selected from the group consisting of lysine, or derivatives thereof, diamine compounds, triamine compounds or tetraamine compounds;
The repeat unit is selected from the group consisting of amidoamine, lysine or lysine analogs;
The surface building unit may be the same as or different from that of the building unit and is selected from amidoamine, lysine or lysine analogues;
Capping group 1 is the residue of a pharmaceutically active agent, derivative thereof, or precursor thereof;
Capping group 2 is selected to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule;
m represents the sum of repeating units of one or more subsurface layers of the dendrimer and is an integer from 1 to 32;
n represents the number of surface building units of one or more surface layers of the dendrimer and is an integer from 2 to 32;
p represents the number of one capping group and is an integer from 1 to 64;
q represents the number of two capping groups and is an integer from 1 to 64]
A lysine dendrimer polymer (where capping group stoichiometry refers to the number and type of capping groups).   24. The dendrimer of claim 21, wherein the polymer further shows a controlled topology (in which case the topology is different from a capping group in terms of its connection to the surface building units or repeating units of the polymer). Represents a relationship with two capping groups).   The dendrimer according to claim 22, wherein the capping group 2 is polyethylene glycol (PEG) or polyethyloxazoline.   23. The polymer according to claim 22, wherein the capping group 2 is a residue of folic acid or a folic acid derivative.   23. The dendrimer of claim 22, wherein the pharmaceutically active agent is selected from the group consisting of methotrexate; taxol; indomethacin; xenical; and cyclosporine. (I) a growing polymer comprising an outer layer having a functional group and two or more different protecting groups,
A precursor of a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer. Supplying a precursor;
(Ii) deprotecting the functional group on the outer layer by removing the first protecting group;
(Iii) activating one of the first or second end group precursors;
(Iv) reacting the deprotected functional group with an activated end group precursor;
(V) deprotecting the functional group on the outer layer by removing the second protecting group;
A controlled end comprising: (vi) activating the other first or second end group precursor; and (iv) reacting the deprotected functional group with the activated end group precursor. Method for producing a polymer with group stoichiometry (in this case, end group stoichiometry refers to the number and type of end groups).   27. The method of claim 26, wherein the growing macromolecule comprises at least one lysine or lysine analog motif.   27. The method of claim 26, wherein the second end group is polyethylene glycol (PEG) or polyethyloxazoline.   27. The method of claim 26, wherein the second terminal group is a residue of folic acid or a folic acid derivative.   27. The method of claim 26, wherein the pharmaceutically active agent is selected from the group consisting of methotrexate; taxol; indomethacin; xenical; and cyclosporine. (I) a growing polymer comprising an outer layer having a functional group and one or more protecting groups, and a carboxylate group,
Surface modifier compound comprising a pharmaceutically active agent, a first end group that is a residue of a derivative or precursor thereof, and a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer Supplying steps,
(Ii) activating a carboxylate group on the surface modifier compound;
(Iii) deprotecting functional groups on the outer layer of the growing polymer by removing protecting groups; and (vii) surface modifying the deprotected functional groups on the growing polymer. A method for producing a polymer with controlled end group stoichiometry, comprising reacting with activated carboxylate groups on the agent compound (where end group stoichiometry is end group stoichiometry) Refers to the number and type of   32. The method of claim 31, wherein the growing macromolecule comprises at least one lysine or lysine analog motif.   32. The method of claim 30, wherein the surface modifier compound comprises a lysine or lysine analog backbone. A method for producing a surface modifier compound comprising:
(i) lysine or lysine analog compounds having two or more different amine protecting groups and carboxylate groups,
A precursor of a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and a second end group selected to modify the pharmacokinetics of the pharmaceutically active agent and / or polymer. Supplying a precursor;
(Ii) deprotecting a primary amine on the protected lysine or lysine analog compound;
(Iii) activating the first or second end group precursor;
(Iv) reacting the activated end group precursor with a deprotected amine group;
(V) deprotecting the secondary amine on the protected lysine or lysine analog compound;
(Vi) activating the other first or second end group precursor, and (vii) reacting said other activated end group precursor with a second deprotected amine group to surface Obtaining a modifier compound.   35. The method of claim 34, wherein the second end group is polyethylene glycol (PEG) or polyethyloxazoline.   35. The method of claim 34, wherein the second terminal group is a residue of folic acid or a folic acid derivative.   35. The method of claim 34, wherein the pharmaceutically active agent is selected from the group consisting of methotrexate; taxol; xenical; and cyclosporine. A surface layer, at least one sub-surface layer, and a first end group that is the residue of a pharmaceutically active agent, derivative or precursor thereof, and to modify the pharmacokinetics of the pharmaceutically active agent and / or macromolecule A polymer with controlled end group stoichiometry comprising at least two end groups including a selected second end group;
A pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient therefor (in this case, end group stoichiometry refers to the number and type of end groups).   39. The pharmaceutical composition according to claim 38, wherein the polymer further exhibits a controlled topology (where the topology is a certain end group in terms of its connection to the surface layer or sub-surface layer of the polymer) Represents the relationship with another terminal group).   39. The pharmaceutical composition according to claim 38, wherein the second end group is polyethylene glycol (PEG) or polyethyloxazoline.   39. The pharmaceutical composition according to claim 38, wherein the second terminal group is a residue of folic acid or a folic acid derivative.   40. The pharmaceutical composition of claim 38, wherein the pharmaceutically active agent is selected from the group consisting of methotrexate; taxol; xenical; and cyclosporine. A method for treating a tumor in a patient in need of tumor treatment comprising:
A surface layer and at least one sub-surface layer, and a first terminal group that is the residue of an antitumor pharmaceutically active agent, derivative or precursor thereof,
A polymer with controlled end group stoichiometry, comprising a second end group selected to modify the pharmacokinetics of the anti-tumor pharmaceutical agent and / or polymer;
A method comprising administering an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient therefor (in this case, end group stoichiometry is the number and type of end groups Point).   44. The method of claim 43, wherein the antitumor pharmaceutically active agent is selected from the group consisting of methotrexate; taxol; cisplatin; carboplatin; and doxorubicin.   44. The method of claim 43, wherein the second end group is polyethylene glycol (PEG) or polyethyloxazoline.   44. The method of claim 43, wherein the second terminal group is a residue of folic acid or a folic acid derivative. A method for targeted delivery of a pharmaceutically active agent to an animal's lymphatic system, comprising:
A surface layer, at least one sub-surface layer, and a first terminal group that is the residue of a pharmaceutically active agent, derivative or precursor thereof,
An effective amount of a polymer with controlled end group stoichiometry, including a second end group selected to facilitate uptake of the pharmaceutically active polymer into the lymph;
A method comprising administering a pharmaceutically acceptable carrier, diluent or excipient therefor (wherein end group stoichiometry refers to the number and type of end groups).   48. The method of claim 47, wherein the second end group is polyethylene glycol (PEG) or polyethyloxazoline.   49. The method of claim 48, wherein the second end group is PEG with an average molecular weight greater than about 1000 daltons.

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